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BOOKS IN SOILS, PLANTS, AND THE ENVIRONMENT

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Agricultural Engineering Robert M. Peart, University of Florida, Gainesville


Animal Science Harold Hafs, Rutgers University,New Brunswick,
New Jersey
crops Mohammad Pessarakli, University of Arizona,
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Irrigation and Hydrology Donald R. Nielsen, University of California, Davis
Microbiology Jan Dirk van Elsas, Research Institute for Plant
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Soils Jean-Marc Bollag, Pennsylvania State University,
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Soil Biochemistry, Volume I , edited by A. D. McLaren and G. H. Peterson


Soil Biochemistry, Volume 2, edited by A. D. McLaren and J. SkujinS
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Organic Chemicals in the Soil Environment, Volumes I and 2, edited by C .


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Humic Substances in the Environment, M. Schnitzer and S. U.Khan
Microbial Life in the Soil: An Introduction, T. Hattori
Principles of Soil Chemistry, Kim H. Tan
Soil Analysis: Instrumental Techniques and Related Procedures, edited by
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SoilReclamationProcesses:MicrobiologicalAnalysesandApplications,
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Symbiotic Nitrogen Fixation Technology, edited by Gerald H. Elkan
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Principles of Soil Chemistry: Second Edition,Kim H. Tan
Water Now in Soils, edited by Tsuyoshi Miyazaki
Handbook of Plant and Crop Stress, edited by Mohammad Pessarakli
Genetic Improvement of Field Crops, edited by GustavoA. Slafer
Agricultural Field Experiments: Design and Analysis, Roger G. Petersen
Environmental Soil Science, Kim H. Tan
Mechanisms of Plant Growthand Improved Productivity: Modern Ap-
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Selenium in the Environment, edited by W. T. Frankenberger, Jr., and Sally
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Plant-Environment Interactions, edited by Robert E. Wilkinson
Handbook of Plant and Crop Physiology, edited by Mohammad Pessarakli
Handbook of Phytoalexin Metabolism and Action, edited by M. Daniel and R.
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fungal Pathogenesis in PlantsandCrops:MolecularBiologyandHost
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and R. Bruce Curry
Agricultural Biotechnology, editedby Arie Altman
Plant-Microbe Interactions and Biological Control, edited by Greg J . Boland
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Handbook of SoilConditioners:SubstancesThatEnhancethePhysical
Properties of Soil,edited by Arthur Wallace and Richard E. Terry
Environmental Chemistry of Selenium, edited by William T . Frankenberger,
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Principles of Soil Chemistry: Third Edition, Revised and Expanded, Kim H.
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and Radhey La1 Kushwaha
Mycotoxins in Agriculture and Food Safety, edited by Kaushal K . Sinha and
Deepak Bhatnagar
Plant Amino Acids: Biochemistry and Biotechnology,edited by Bijay K . Singh
Handbook of functional Plant Ecology, edited by Francisco I. Pugnaire and
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HandbookofPlantandCropStress:SecondEdition,RevisedandEx-
panded, edited by Mohammad Pessarakli
Plant Responses to Environmental Stresses: From Phytohormones to Ge-
nome Reorganization, edited by H. R. Lerner
Handbook of Pest Management, editedby John R . Ruberson
Environmental Soil Science: Second Edition, Revised and Expanded,Kim H.
Tan
Microbial Endophytes, edited by Charles W . Bacon and James F. White, J r .
Plant-EnvironmentInteractions:SecondEdition, edited by RobertE. WiI-
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Microbial Pest Control, Sushi1 K. Khetan
SoilandEnvironmentalAnalysis:PhysicalMethods,SecondEdition,Re-
vised and Expanded, editedby Keith A. Smithand Chris E. Mullins
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WoodyPlantsandWoodyPlantManagement:Ecology,Safety,andEn-
vironmental Impact, Rodney W. Bovey
Handbook of Postharvest Technology, A. Chakraverty, Arun S. Mujumdar,
and G.S.V. Raghavan
Metals in the Environment M. N. V. Prasad
This Page Intentionally Left Blank
MICROBIAL
PEST
CONTROL
Sushi1 I<. I<hetan
Crop Protection Technologies Consultant
Pittsburgh, Pennsylvania

M A R C E L

MARCELDEKKER,
INC. -
NEWYORK BASEL
D E K K E R
Library of Congress Cataloging-in-Publication Data

Khetan, Sushil K.
Microbial pest control 1 Sushil K. Khetan.
p. cm. -- (Books I n soils, plants, and the envlronment ; v. 78)
ISBN 0-8247-0445-2 (alk. paper)
1. Pests--Biological control. 2. Microbial toxins. 3 . Agricultural microbiology. 4.
Microorganisms. I.Title. 11. Series.

SB975 .K54 2000


632'.96--dc21

00-060208

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Foreword

Regulation of pesticides in the U S . is a dynamic process being influenced from


time to time by acts of Congress changing the basic laws. Significant changes were
made in 1980 in the regulation and safety testing of naturally occurring pesticides
("biorational" pesticides). The United States Environmental Protection Agency
(EPA) considers biochemical pest control agents, microbial pest control agents and
microorganisms as biorational pesticides under the Federal Insecticide, Fungicide
and Rodenticide Act (FIFRA), but its policy is to regulate only biochemical and
microbial pest control agents. The Agency requires three types of data
requirements-product analysis, hazard to humans and domestic animals
(toxicology), and hazard to nontarget organisms-for the registration of biochemical
and microbial pesticides. Several biopesticides, including microbial pesticides, have
since been registered using these criteria.
Microbial pesticides have gained prominence in recent years for control
of plant pests and diseases. The regulatory agencies have recognized these
products, among others, as alternatives to conventional chemical pesticides
which are known toshow persistence in environmental media, lack specific
mode of action, and leavetoxic residues that affect humanhealthand
environmental safety. With the emergence of new technologies in agriculture,
several new varieties of microbial products were developed and, especially
during the last 5 years or so, the whole field of microbial pesticideshas
exploded.
Biotechnology is a broad term that encompasses a wide spectrum of
applications used traditionally in the past to the rapidly advancing cutting edge
technologies. It has had a profound impact on the food and agriculture industry.
Some proponents of biotechnologysee it asa way to greatly increase
agricultural production while minimizing environmental damage. Some critics
view biotechnology as causing devastation to the natural environment that will
ultimately corrupt our ability to produce food. Lost in this exchange is the fact
that biotechnology is a multifaceted spectrum of technological developments.
Biotechnology is currently the most powerful tool to further advance
the various fields of agriculture. However, it can work practically only if it can
be combined with established breeding strategies and with common agricultural
practices. With the human population expected to reach 10 billion by the year
2050, the challenges facing agriculture andfood research areenormous.
Therefore, there is an urgent need to intensify our development of agricultural
biotechnologies.
Transgenic crops are created by implanting specific gene sequences from
a donor species with desired traits into a host plant to produce a new variety
expressing the traits of the donor organism. For example, the gene sequences
...
111
iv Foreword

controlling the production of crystal endotoxins in Bacillus thuringiensis (Bt)


bacteria have been transferred to cotton, corn, and potato plants to provide plant-
wide insect resistance.
Many of the currently developed transgenic crops can, at least in the
short term, enhance economic returns and decrease environmental impacts of
agriculture. Whether this ultimately increases the sustainability of agriculture
dependson whether the economicgainsand environmental improvements
continue in the future. Reduced reliance on chemical pesticides can benefit the
surrounding ecology and the health of agricultural workers and consumers of
agricultural products. However, if transgenic crops result in the development of
resistant pest populations, the benefits of technology will disappear over time
and may lead to increased chemical costs or reduced crop yields in the future.
Similarly, transgenic crops that result in the creation of new weeds or diseases
will not be sustainable.
The most widespread application of genetic engineering in agriculture
by far is in engineered crops. Thousands of such products have been field tested
and over a dozen have beenapproved for commercial use. The traits most
commonly introduced into crops are herbicide tolerance, insect tolerance, and
virus tolerance.
All of the commercially available insect-tolerant plants contain a version
of the toxin Bt. A major concern is that wide use of Bt crops will lead to resistance
to the toxin. If resistance develops, the Bt toxin will be useless as a pesticide. In
such a case, the environmental benefits of the product will be short lived.
The EPA oversees genetically engineered microbial pesticides and
certain genetically engineered crops under FIFRA. Thereappearsto be two
major concerns-pest resistance and safety of non-target insects-for transgenic
crops such as transgenic cotton and corn, which have been approved for use in
the U.S.These transgenic plants are developed by inserting genes from
microbial pesticides that are known to be safe to humans and animals and donot
harm beneficial insects. The EPA and USDA have been working with the
industry to develop pest resistance management plans to avoid the build-up of
resistance.
Another concern is the safety of the non-target organisms. In December
1999, EPA issued a notice to registrants of Br corn plant-pesticides requiring
submission of data on the effects of Bt corn pollen on non-target species,
particularly the Monarchbutterfly.This arose out of a preliminary study
conducted by Cornel1 University suggesting that pollen from corn genetically-
enhanced to express the Bt toxin may pose risks to Monarch butterfly larvae. To
evaluate whether Br corn pollen presents risks to the Monarch butterfly and
other non-target lepidopteran species, the Agency requires extensive data
including toxicity testing on the species of butterfly, milkweed distribution, and
pollen dispersal and the development of appropriate models to test the risks to
non-target insects in Bt corn fields.
Foreword V

Consumer acceptance is an important component of economic success


ofthebiotechnology. If consumersdecide that they preferproducts without
utilization of the genetically altered crops, then the producers of the genetically
altered crops will face an uphill battle. Consumer perceptions of the risks and
benefitsassociated with productsderivedfrombiotechnologyappear to be
driven by attitudes linked with specific applications of genetic engineering. It is
essential to make the public
understand the risks versus benefits of
biotechnology.
In any country, there are people who want to apply new technologies
including biotechnology in agriculture, and those who do not. The issues related
to agricultural biotechnology seemto go deeper than religion or culture and they
will always bedivided. Bioethicsaddressesthe ethical issues in biologyand
medicine using biotechnology. These ethical issues arenot used to prescribe the
correct answers but to make good choices for improving our life and society.
The bioethical principles for agricultural biotechnology should consider, among
many others, autonomy of choice versus justice, balancing benefits and risks,
ethical values in life and nature, and sustainability and balancingideals.
Scientists should play a major role in bioethics of biotechnology and the public
should be informed of the ethical, environmental and social issues that science
raises.
The United States rapidly
is moving in
commercialization of
geneticallyengineeredproductsforcropproduction and cropprotection.
Introduced only a few years ago, the transgenic crops (cotton, corn and others)
have already made significant inroads for agricultural biotechnology in the U.S.
Potential markets are big; however, there are several unforeseen problems such
as pest resistance, gene transfer to wild plants and unknown harms that may be
realized
over
a long period. So long as the benefits from the new
biotechnologies outweigh the potential risks, the U.S. is likely to promote the
use of thesenewtechnologiesforimprovedcrop yields andreduced use of
pesticides and fertilizers,amongothereconomic,social and environmental
benefits.
This book examines the state of the art, science and technology for the
development,productlon, modeand mechanlsm of action, and applicationof
bacterial and viral insecticides, biofungicides, bioherbicides
and
mycoinsecticides. Dr. Khetan has made an attempt to give an overview of the
integrated uses of microbial pesticides with conventional chemical pesticides,
and commercialization of nlicrobial pesticides. Publication of this book seems
timely when the regulators and the public are scrutinizing the transgenic crops
andthe genetically modified (GM) foods, while the industry is tryingto
convincethemthat GM foodsareassafeasconventional foodsand that
transgenic crops would surely contribute to solving the global food problem in
this century. This book will educate those who want to understand what these
microbial pesticides are as well as the implications of their application to crop
vi Foreword

protection. Dr. Khetan should be commendedfor summing up the information in


this field at this critical juncture.

N. Bhushan Mandava, Ph.D.


President
Mandava Associates
Preface

Growing concern for safetyof human health and the environment has pushed the
regulatory process to screen and control toxicants at levels that are perceived
safe.There is growing worldwide regulatory effort topromote biologically
based pest control that includes botanicals, biochemicals and microbials. Among
these, microbial pestcontrolagents as agroup have seenrapid advances in
recent years. The discovery and development of new microbial control agents
and technologies capable of enhancing efficacy have been largely responsible
for this. Genetic improvements of microbials using both recombinant and non-
recombinant methods and compatibility with other interventions have resulted in
a number of commercial products. The development and commercial availability
of natural epizootics of fungi and viruses have expanded these options. The idea
of using one microorganism against another, the predator versus the prey, offers
an environmentally compatible and intellectually appealingapproach for the
control of plant diseases. Similarly, phytopathogenic fungi and bacteria have
shown promise in weed management. A synopsis of the literature on the current
uses of bacteria, viruses, and fungi as microbial controlagents along with
insights on their potential are presented in this book.
Microbials are not envisaged to replace chemical pesticides on major
crops, such ascotton, corn, wheat and rice in the near future However,
microbials are viewed as a key component of integrated pest management (IPM)
programs, as supplements to chemical pesticides in combination or in
succession. Selectivityand minimal environmentalimpact of microbials are
desirable features for IPM programs. Also if they were used as direct
replacements of chemical pesticides, then eventually these agents would face
some of the same fate as the chemicals they replace, particularly with respect to
resistance. Nevertheless, exclusive use of biopesticides may have niche markets
oncrops such ashigh-value fruits, nuts, vegetables and ornamentals. This is
either asaconsequence of some regulatory action or for tapping a specific
activity unattainable through chemical pesticides.
There is a wealth of information on the state of the art and the level of
commercialization reached among microbials for pest control. However, most of
this is difficult to retrieve, being scattered in various discipline-based literature
and Internet-accessible databases. Information retrieval of this nature also
requires training inseveral specialized areas.The traditional chemical-based
pest control products industry has long years of experience in developing and
commercializing products for the plant protection market. However, the
industrys technical pool, which consists of chemists and chemical engineers as
well as entomologists and plant pathologists, faces difficulties in accessing and
fully comprehending these new developments. On the other hand, the nascent

vi i
viii Preface

small biologically-based pest control industry has struggled in devising product


handling and commercialization strategies. These
limitations may have
constrained the growth and commercialization of biologically-based pest control
agents. End-use based products integration has been in the offing within large
chemical companies by forming life sciences divisions. However, medium and
small chemical companies have been left out as they do not have the information
resources of large companies, and have often been at a loss to figure out their
areas of opportunity. There is an apparent need for a viable way for chemical
and other specialty professionals for gathering first-hand knowledge of
emerging microbial control systems and their potential.
My aim in writing this book was to bring together the latest advances in
the science and technology of microbial pest control and in the evolving field of
biopesticides. However this is not intended to be a comprehensive treatise. My
effort is to presentstate of art information to the non-specialist reader in a
manner the is easy and interesting. The approach is to facilitate understanding of
this complex subject across discipline boundaries. A glossary of technical terms
has also been prepared with this conslderation. To aid rapid understanding of the
material, numerous figures, drawings and chemical equations have been used. I
hope these would also find use as teaching and presentation aids.
This book offers an overview of technologies that are driving the rapid
proliferation of microbial based pest control. It alsoprovides coverage of
products that have reached the marketplace. Promising strategies for the
development of effective biological controls for plant and vector borne pests,
plant diseases and weed management are discussed by addressing many of the
critical issues. Several current topics such as genetically altered Bacillus
thuringiensis ( B t ) and transgenic crops, microbial formulations and synergistic
interactions of microbials with synthetic chemicalshavebeen documented.
Similarly, critical summaries on resistance management of Br foliar applications
and Br genes in transgenic crops have beenprovided. Current states of
technologies of viral and fungal insecticides and bacterial and fungal
biofungicides and bioherbicides have beendiscussed. An analysis of related
technical, social and economic issues that govern the commercialization efforts
of biopesticides is also provided.
The book is divided Into three parts consisting of eleven chapters. The
first part covers various aspects of the Br bacterium exclusively and consists of
five chapters. Bt is the major success story of microbial pest control. The first
chapter provides a comprehensive review of Br delta-endotoxins. It also includes
their mode of action as bioinsecticides and safety to non-target organisms and
environment. The second chapter discusses Br subspecies specific for control of
caterpillar pests in agriculture and for control of mosquitoes that act as carriers
of disease. A discussion on Bacillus sphaericus has been Included, enumerating
its growing commercial importance in vector control. Chapter Three covers
various products based on genetically modified Bt as well as on transgenic Bt
Preface ix

organisms and Bt plants. In Chapter Four, bacterial formulations including


various efficacy-enhancing strategies are discussed. Various formulation types
specific to Bt and B. sphaericus and their advantages are also covered. Chapter
Five deals with resistance development in insect target populations towards Bt
toxins by spray applications and through transgenic Bt plants. Resistance
management strategies are also covered in this chapter.
Part Two consists of four chapters. Chapter Six discusses natural and
recombinant viral bioinsecticides and the progress made in the use of
baculoviruses for enhanced virulance and stability. Biofungicides are discussed
in Chapter Seven, which covers fungal and bacterial antagonists, including their
modes of action, anddescribes various commercial products.Fungal and
bacterial pathogens used asbioherbicides are discussed inChapter Eight.
Chapter Nine on mycoinsecticides reviews the progress made in
commercialization of entomopathogenic fungi. The third part consists of two
chapters. Chapter Ten provides an overview of integrated use of biopesticides
and use with synthetic pesticides. Several examples areprovided here to
demonstrate synergistic interactions of various combinations for reliable and
consistent control of pests, weeds and diseases. The concluding Chapter Eleven
discusses the issues involved in commercialization of biopesticidal products. It
enumerates approaches employed for successful commercialization citing case
studies from the literature.
It is hoped that industry managers, regulatory personnel and
environmental safety and health planners and all those with an interest in safer
approaches to pest control, will find the book a usefd resource. This
presentation will be found
equally useful by crop and soil scientists,
entomologists, plant pathologists, mycologists, microbiologists, formulation
technologists, and biochemical engineers. As the field has grown rapidly, it may
also provide useful overview of the state of art for researchers who are working
on any single aspect of microbial pest control. It is my hope that the material
contained here will lead to abetter appreciation of microbial pest control
strategies stimulating further research, production and commercialization of
biopesticides.
Evolving sciences often progress by taking two steps forward and one
stepbackward.The ongoing controversy and intense speculation about
AgBioTech industry are, perhaps, a manifestation of this axiom. It is observed
that a market for a product exists based onthe consumers perception of it. Their
level of awareness of the product shapes this perception. If this book enhances
the level of awareness of microbial pest control by providing asmallstep
towards understanding of many new developments, my objective in writing this
book will have been achieved.
X Preface

Acknowledgements

I acknowledgea large numberofacademicandindustry scientists and


technologists for their support. Particularly, I would like to thank Greg Boland
(UniversityofGuelph), Teny Couch(BeckerMicrobials),BrianFederici
(University of California, Davies), Kunthala Jayraman (Anna University), N.
Bhushan Mandava (Mandava Associates), Bineeta Sen (IARI, New Delhi) and
Anurag Khetan (Merck & Co.) for offering their critical comments and useful
suggestionsduringvariousstagesofthe writing. I acknowledgeAjayGupta
(Hindustan Insecticides), a valued colleague, who has provided active support
throughout the preparationofthemanuscript.Hiscontributionstowards
literature search, preparation of review chapters on Bt, preparation of indices
and proofreading of the manuscript, have been of immeasurable help. I also
gratefully acknowledge the timeandorganizationalsupportprovidedby the
Chairman and Managing Director, Hindustan Insecticides Limited, New Delhi
in completing a major part of this book.
AshishKhetan(CarnegieMellonUniversity)provided significant
technical support in the preparation of this manuscript and camera-ready copy.
Kishan La1 did the initial typing. I would also like to thank the production team
at Marcel Dekker,Inc., for providing consistent support and timely follow-up.
Finally, I would like to acknowledge the constant support of my wife
Manju, who endured the endlesshours of assimilating the informationand
putting it together in the manuscript. Without her patience and understanding
this venture would not have been possible.

Sushi1 K. Khetan, Ph.D.


Contents
...
Foreword by N. Bhushan Mandava 111

Preface vii

Part I. BacterialInsecticides

Chapter 1. Bacterial
Insecticide: Bacillus
thuringiensis 3

Introduction 3
Production of Bt 9
Classification and Nomenclature of
Bt 13
Bt Crystal Proteins and Genes 16
Insecticidal Activity 21
Mode of Action 24
Persistence 30
Safety and Ecotoxicologyof Bt 32
Concluding Remarks 34

Chapter2.BacterialInsecticidesforCrop and Forest


Protection and Insect Vector Control 43

Introduction
1 43
2 Bacillusthuringiensis subsp. kurstaki 44
3 Bacillusthuringiensis subsp. israelensis 54
4 Bacillus
sphaericus 60
5 Concluding Remarks 69

Chapter 3. Genetically
Modified Bt Strains and
Bt Transgenic Plants 77

1 Introduction 77
2Novel Bt StrainsThroughConventional
Genetic Techniques 78
3RecombinantDNATechnology 81
4Insect-TolerantTransgenicCropPlants 89
5 Concluding Remarks 95

xi
xii Contents

Chapter 4. Formulation of Bacterial Insecticides 101

Introduction 101
Characteristics of Microbial Insecticide
Formulations 102
Formulation Attributes Having Impact on
Efficacy of Bacterial Insecticides 103
Commonly Used Formulationsof Bt 111
Improved Bt Formulations 112
Target-Specific Tailor-Made Formulations
of Bacterial Larvicides 116
Efficient Delivery is Equivalent to Effective-
ness at Low Dose 118
Concluding Remarks 118

Chapter 5 . Insect Resistance to Bt Toxins 123

1 Introduction 123
2 Resistance to Bt toxins in Lepidoptera 124
3 Synergistic Interactions of Bt-israelensis
Toxins and Effect on Resistance Development 130
4 Insect Resistance to B.sphaericus 132
5 Resistance Management of Bt Toxins 132
6 Concluding Remarks 140

Part 11. Viral Insecticides, Biofungicides, Bioherbicides,


and Mycoinsecticides

Chapter 6. Natural and Recombinant Viral Insecticides 147

1 Introduction 147
2 Baculovlruses 147
3 Genetically Modified Baculoviruses
for Insect Control 157
4 Concluding Remarks 163

Chapter
Biohngicides
7. 167

1 Introduction 167
2 Biological Control of Soil-Borne Diseases 169
3 Soil-borne Diseases 176
Contents xiii

4 Biological Controlof Aerial Plant Diseases 181


5 Control of Post-Harvest Decay of Fruits
and Vegetables 184
6 Biological Control with Trichoderma Species 186
7 Biological Control with Fluorescent
Pseudomonads 189
8 Production of Biofungicides 190
9 Formulation of Biofungicides 191
10 Concluding Remarks 193

Chapter 8. Bioherbicides 199

Introduction 199
Fungal Bioherbicides 200
Bacterial Bioherbicides 203
Environmental Limitations for Efficacy
of Bioherbicides 204
Production of Bioherbicides 205
Formulation of Fungal Bioherbicides 205
Formulation of Bacterial Bioherbicides 208
Concluding Remarks 208

Chapter 9. Mycoinsecticides 21 1

Introduction
1 21 1
2ModeofAction 213
3Production of Mycoinsecticides 215
4Formulation of Mycoinsecticides 217
5 Concluding Remarks 22 1

Part 111. Integrated Use and Commercialization of Biopesticides

Chapter10. Integrated Use of Biopesticides and Synthetic


Chemical Pesticides 225

1 Introduction 225
2IntegratedUse of Biofungicides 226
3IntegratedUseofBioherbicides 23 1
4 IntegratedUse ofBioinsecticides 234
5 Concluding Remarks 240
xiv Contents

Chapter 1 1. CommercializationofBiopesticides 245

1 Introduction 245
Commercialization
2 Aspects 247
3 Commercializationof Biofkgicides 250
4Commercialization of Bioherbicides 252
5CommercializationofBioinsecticides 254
6 Commercialization of Pest-ResistantCrops 257
Concluding
7 Remarks 261

Glossary, and Product, Manufacturer, Pathogen and


Subject Indices 265

Glossary 267
Product Index 277
Manufacturer Index 283
Pest and Pathogen Index 28 7
Subject Index 295
Part I

Bacterial Insecticides
This Page Intentionally Left Blank
Bacterial Insecticide:
Bacillus thuringiensis

I Introduction
The critical need for safe and effective alternatives to chemical insecticides has
stimulated considerable interest in using pathogens as biological control agents
for insects of agricultural and medical importance. Both chemical and biological
insecticides are currently used for insect control but the use of biological
insecticides is favorable because they kill undesirable agricultural and household
pests and vectors of human and animal diseases without introducing toxic and
non-biodegradable substances into the ecosystem.
Microbial pesticides are becoming recognized as an important factor in
crop and forest protection and in insect vector control. These pesticides are
naturals, disease causing microorganisms suchas viruses, bacteria and fungi,
that infect or intoxicate specific pest groups. The pathogen that has been the
most successhl and holds considerable potential for further development is the
insecticidal bacterium Bacillus thuringiensis (Bt). Otherbacterial insecticides
exist, but have not yet achieved the commercial success of Bt.
Bt-based insecticides provide growers and the public with
environmentally friendly and effective alternatives to conventional insecticides.
These are (a) not harmful to natural enemies and other non-target organisms due
to narrow host specificity, (b) harmless to vertebrates including mammals,
3
4 Chapter 1

(c) biodegradable inthe environment and (d) highly amenable to increase in


activity by genetic engineering.
A number of excellent reviews have appeared in the literature on the
subject (Schnepf etal., 1998, Prieto-Samsonov et al.,1997; Cannon, 1996;
Kumar et al., 1996; Koziel et al., 1993; Beegle and Yamamoto 1992; Lambert
and Peferoen 1992 and Peferoen, 1992).

1.1 History

The first record on Bt goes back to 1901, when Ishiwata discovered a bacterium
from diseased silkworm larvae that he named Bacillus sotto. Berliner isolated a
similar bacterium from Mediterranean flour moth (Anagasta kuekniella) in
1911. The bacterium was named Bacillus thuringiensis after Thuringia,
Germany, where the infected insect was found.
Husz isolated a Bt strain from Ephestin in 1928, tested it on European
corn borer and reported the first application of Bt. This work eventually led to
the first commercial product, Sporeine, which was produced in France in 1938.
The development of potent organic insecticides, however prevented rapid
advancement in the development of biological alternatives for pest control. The
pioneering research of Steinhaus (195 1) on Bt and a growing realization that
organic insecticides are deleterious to the environmentandhumanhealth
spurred renewed interest in Bt. This led to the introduction of aviable Bt
biopesticide called Thuricide in 1957, a product name still in use.
During 1960s, several commercial formulations of Bt were
manufactured in the United States, Soviet Union, France, and Germany andused
with various degrees of commercial success. A major advance occurred when
Howard Dulmage and Clayton Beegle of the USDA AgriculturalResearch
Service assembled the first collection of Bt strains. Dulmage isolated the HD-1
strain, which is still used as a reference in many studies and is included in many
Bt-based products (Dulmage, 1970).
Until the 1970s, itwas generally believed that Bt was onlyactive
against Lepidoptera. The discovery of Bt subsp. israelensis, which is toxic to
larval mosquitoes and blackflies (Goldbergand Margalit, 1977) and the
discovery of Bt subsp. tenebrionis (Krieg et al., 1983), which is toxic to several
beetle species, stimulated sharply increased commercial interest in Bt. During
the 1980s, it led to foundation of new biotech companies whose core business
consisted of the development of novel Bt-based insecticides. By 1992, it was
estimated that 40,000 strains of Bf were stored, mainly in private collection
worldwide. Now,numerous commercial products formulated with Bt are
registered for control of larvae of lepidopterous insects (moths and butterflies),
dipterous insects (flies and mosquitoes) and coleopterous insects (beetles).
Bacillus 5

1.2 Occurrence

Bt is ubiquitous in soils and the foliar surface (phylloplane). These habitats must
inadditionto insect larvae, constitute viable niches of Bt. Indeed, Bt is a
facultative pathogen, in contrast to B. popilliae for example, which is strictly
parasitic and cannot live independent of its host.
Bt has a worldwide distribution in various soils, including forest soils,
savannas and deserts and in insect rich environments such as sericulture farms,
insect-rearing facilities, grain dust from flour mills, and grain storage facilities.
The environmental distribution of Bt is not related to the distribution of the
target insect(s). Nevertheless, certain Bt serotype appear to accumulate in the
environments where insects are abundant andor breeding (Delucca et al., 1981;
Bernhard et al., 1997 and Chaufaux et al., 1997).
Bt can be isolated from a wide variety of habitats. It is readily found in
cadavers of insects from stored product dusts than from soil, but it doesnt grow
well in many of these because it is not a major or dominant species (Bernhard et
al., 1997; Chaufaux et al., 1997). Moreover, itis interesting that, unlike many
viruses, fungi and protozoa, Bt has never been reported as the cause of large-
scale epizootics. Despite Bts wide spread occurrence in nature, optimal
reproduction probably occur in insects (Federici, 1993).
Bt is easily grown and completes its growthcycleonordinary
laboratory media with aminimum of nutrients. It is, however, incorrect to
assume that Bt germinates, grows and multiplies in nature if appropriate
nutrients are available. Although Bt spores may survive for many years, they do
not germinate and multiply as vegetative cells in natural soils. This observation
indicates that Bt is not adapted to the soil environment. Under natural
conditions, crystal proteins released from the spores into soilare rapidly
degraded, but spores are more long lived. Although these sporeshavesome
toxic activity, extremely high numbers of sporesare required to kill larvae.
Therefore, given the low number of Bt spores, this bacterium has probably no
significance in the control of the insect populations in natural environments and
is unable to multiply in this environment (Saleh et al., 1970 and Lambert and
Peferoen, 1992).

1.3 Characteristics

Bacillus thuringiensis (Bt) is a gram-positive, rod shaped, aerobic, endospore


forming, ubiquitous soil bacterium that belongs to morphological group I, along
with Bacillus cereus, Bacillus anthralis and Bacillus laterosporus.
Bt produces a parasporal body within its cells during sporulation. This
parasporal body contains one or more proteins, typically in a crystalline form
6 Chapter 1

0
Figure 1.1 Photomicrograph of Bacillus thuringiensis bacteria.The white
spotsinside the bacteria are the protein crystals(Courtesy
Monsanto Co.).

(Figure 1.1). These proteins possess insecticidal properties and are called Bt
toxins, &endotoxins or insecticidal crystal proteins (ICPs). Insecticidal crystal
proteins produced by Bt, kill insects by binding to and disrupting their midgut
epitheliummembranes,yetdoeslittleornoharm to most other organisms,
including human,wildlife and other insects.
Bt producesalargevarietyofinsecticidalcrystalproteins,which
reflects its remarkable adaptive capacity to settle in the large ecological niche
constituted by insects. The biological mechanisms used by the bacterium to
make enough toxin to kill insects are a striking example of bacterial evolution.
Theinsecticidalproteins are synthesizedduringthestationaryphase and
accumulate in the mother cell as a crystal inclusion which account for20 to 30%
of the dry weight of the sporulated celland mainly consists of protein (95%) and
asmallamountofcarbohydrate (5%). Theamountofthecrystalprotein
produced by a Bt culture in laboratory conditions (about0.5 mg of proteidml)
and the size of the crystal indicate that each cell synthesizes lo6 to 2x106 6-
endotoxin molecules during the stationary phase to form a crystal. This would
require
substantial
a dedicated
usage of cell machinery.
Nevertheless,
sporulation and the associated physiological changes also proceed in parallel
with &endotoxin production (Agaisse and Lereclus, 1995). The &endotoxins,
which are deposited in the cytoplasm of thebacterium, are ofincreasing
importance as biopesticides. In this respect, Bt represents an intermediate
between biocontroland synthetic insecticides, with role a restricted to thatof an
ingestion toxicant (Cannon, 1996).
Bacillus thuringiensis 7

cell

Germination

Figure 1.2 Schematlc representation of a sporulatmg Bacillus thuringiensis


(Adapted from Lambert and Peferoen, 1992).

1.3.1 Life cycle

Bt has two growthphases,germination(vegetativegrowth)andsporulation


(Figure 1.2). During the vegetative cell cycle, when the bacteria are in nutrient
richenvironment, Bt normallymultipliesexponentially bycelldivision.
Sporulationoccurswhennutrientsaredepletedorwhentheenvironment
become adverse, forming spores (called endospores) within a sporangium. These
sporesarehighlyresistanttoadverseconditionssuchasheatanddrought,
enabling the bacterium to survive periods of stress. Spores germinate and may
restart a vegetative cell cycle under favorable conditions (Lambert and Peferoen
1992 and Itoua-Apoyoloet al., 1995).

1.3.2 Toxinsproduced by Bt

Bt produces mainly &endotoxins and a wide variety of secondary metabolites,


someofwhichareinsecticidal e.g. theheatstableadeninenucleotide, p-
exotoxin and immune inhibitor A (protease), several phospholipases and a heat-
labile a-exotoxin, .lecithinase C. More recent characterization has shown that
chitinases (Sonngay and Panbangred, 1997), zwittermicin (Stabb et al., 1994)
which increases the toxicity of Cry proteins by unknown mechanism (Federici,
1998), andthesecretedvegetativeinsecticidalproteins may contributeto
virulence (Estruch etal., 1996).
8 Chapter 1

a) &EndotoxinThe insecticidal crystal preoteins (ICPs) or &endotoxinsare


a family of insecticidal proteins produced by Bt duringsporulation.The S-
endotoxins have relative molecular masses of 60-70 kDa in the active form and
specific toxicities against insects in the order of Lepidoptera, Diptera and
Coleoptera. These toxins have been formulated into commercial insecticides for
three decadesand now insect-resistant plants are engineered by the
transformation with Lepidoptera-specific toxin genes. Numerous strains of Bt
are currently known. Each strain produces differing numbers of &endotoxins
with various insecticidal activities. A given &endotoxin is typically insecticidal
towards a narrow spectrum of insect targets.

b) p-Exotoxin: Several strains of Bt producelow molecular weight heat


stable insecticidal toxin known as p-exotoxin during vegetative growth. p-
exotoxin or thuringiensen (C22H32N5019PI. 3 H20) is water soluble, dialyzable
nucleotide composed of adenosine, glucose and allaric acid (Figure 1.3). It is
found that the phosphate group on allaric acid is essential for activity and that
the toxicity of p-exotoxin results from its inhibition of RNA biosysthesis by
competing with ATP for binding sites. Some of the Bt subsp. that produce p-
exotoxin are canadensis, ciarmastaciiensis,kumamotoensis, galleriae, nzowisoni,
thuringiensis etc.
Two p-exotoxin containing products are produced and used in Russia,
Thuringin 1 and 2 (2% and 10% p-exotoxin respectively) andBiotoxibacillin
(0.6 - 0.8% p-exotoxin). The products are used effectively againstseveral
species of red mites, as well as against larvae of houseflies and blowflies. Other
products are Dibeta (Abbott labs.) and Exobac (CRC).Current registration
standards for Bt based insecticides requires an absence of p-exotoxin (Cambell
et al., 1987 and Prieto-Samsonov et al., 1997).

Figure 1.3 13-Exotoxin.


Bacillus thuringiensis 9

c) a-Exotoxin: Many strains of thuringiensis


B. produce heat-labile
insecticidal exotoxin known as a-exotoxin and found to be toxic to wax moth
larvae. a-exotoxin is also toxic to insect and mice upon injection. a-Exotoxin
extracted in water from the Thuricide powder, a commercial preparation of Bt,
was toxic to sawfly larvae fed on treated foliage. The estimated size of a-
exotoxin was 45-50 kDa (Beegle and Yamamoto, 1992). Some of the Bt
subspecies that produce a-exotoxin are aizawai,alesti,finitinus,kenyae,
kurstaki, sotto, thompsoni, thuringiensisetc.

d) VegetativeInsecticidalProteins(Vips): The clarified culture supernatant


fluids collected during vegetative (log phase) growth of Bacillus species are a
rich source of insecticidal activities. Vip proteins are distinct from insecticidal
proteins that belong to b-endotoxin family, which are ineffective against
rootworm and cutworms (Nunez-Valdez, 1997).TheseVips do not form
parasporal crystal proteins and are apparently secreted from the cell.
Vips display acute biological activities against major pests of maize,
such as the corn rootworm (Diabrotica spp.) and the black cutworm (Agrotis
ipsilon). Supernatant fluids of certain B. cereus isolates possess acute bioactivity
against Northen and Western corn root worms. The insecticidal properties are
associated with abinary system, whose two proteins are termed ViplA and
Vip2A. Similarly, supernatant fluids of certain Bt cultures have potent
insecticidal properties against black cutworm and fall armyworm. This led to
discovery of Vip3A, a novel insecticidal protein with no homology with any
known protein. This protein is reported to exhibit toxicity towards a wide variety
of lepidopteran insects, including Agrotis ipsilon,
Spodopteraexigua,
Spodoptera frugiperda and Helicoverpa zea (Estruch et al., 1996).

2 Production of Bt
Bacillus thuringiensis has been used to produce biological insecticides for foliar
application for over 50 years. Bioinsecticides derived from B.thuringiensis
contain inclusion bodies, spores, cell debris and other residual solids, which are
all recovered from the broth at the end of the fermentation and then formulated,
packed and dried (Rowe and Margaritis, 1987).
Bioinsecticides were, and are still produced by fermentation of single
Bt strains incrude, inexpensive media. As the Bt cells begin to exhaust the
medium, they enter the sporulation phase of growth where one or more
insecticidal proteins, or 6-endotoxins, are synthesized. The 6-endotoxins
accumulate in morphologically distinct crystal(s) within the cellsand are
released into the medium with the spores when the cell ultimately lyse.
10 Chapter 1

Typically, the first step in the manufacturing process of a Bt bioinsecticide is to


concentrate the fermentationsolids(spores,b-endotoxincrystalsandunused
particulate media components) by centrifugation. Centrifugation or filtration can
recover the suspended solids from the fermented broth of Bt with efficiency
higher than 99% based on spore count (Villafana-Rojas et al., 1996). Sometimes
tangential flow dialysis is also used to further concentrate the solids before spray
drying.Thedried material is thenmilled to auniform size toproducea
technical powder that can be formulated in avariety of ways. The most common
formulations are wettable powders or dispersible granules, but oil and aqueous
flowable preparationshave also been produced.
Three major factors contribute to the effectiveness of Bt insecticides.
First,a Bt strainmustbe obtained that producesb-endotoxinswhichare
effective on targetinsects.Second, the strainmustbeable to produce large
amounts of active &endotoxin during large-scale fermentations at a minimum
cost. Third, a formulation that maximizes the longevity and effectiveness of the
&endotoxins should be used (Kozeil et al., 1993).

2. I Strain Selectiotr

The bacterial strains used for all of the early and some of the current Bt foliar
insecticides are wild type strains, that is, they were found in nature in the form
in which they are used to produce the microbial spray. Most Bt isolates produce
multiple
&-endotoxins, each having characteristic
a insecticidal
activity
spectrum. It is generally believed that the overall activity spectrumof a strain is
due to additive and/or synergistic interspectrum of the individual &endotoxins
present in their proportionalamounts.Themaximumamount of asingle 6-
endotoxin protein would be obtained from strain expressing a singleb-endotoxin
gene at its maximum biosynthetic capacity. However, the insecticidal spectrum
of suchastrain would be relatively narrow. In contrast,astrainproducing
several
6-endotoxins would have broader
a spectrum, but the
activity
contribution of each&endotoxinwouldbedecreased in proportion to the
amount produced.
It is possible to construct strains with increasedactivity on specific
pests byusinggenetic manipulation techniques and geneticengineering to
eliminateunnecessary &-endotoxin andor introducedesirableones. Bt strains
present in severalcurrentlyavailablebioinsecticides were constructedusing
these techniques e.g. Agree (Therm0 Trilogy), Novodor (Abbott), Condor, Foil
and Cutlass (Ecogen) etc. (See Chapter3).
The task of identifyingb-endotoxinswith neworpotentactivities
typically involves screening wild type strain of Bt. When screening for specific
activity, is some times the case that toxicity to a target species is greatest in Bt
Bacillus 11

isolates which commonly occur in the same microbial habitat as the pest. For
example, the majority ofBt isolates collected from silkworm litters were toxic to
silkworm larvae, whereas most isolates from soil collected in Japan showed no
insecticidal activity againstthis species (Koziel etal., 1993).
A large number of Bt isolates are screenedin an attemptto identify new
strainswith increased levels of insecticidalactivityandactivitiesagainsta
broader spectrum ofinsect pests. The discovery rate of novel Bt strains or ICPs
is relatively high, up to 1 in 1000 screened Bts produce a novel ICP compared to
1 in 20,000 screened compounds for synthetic pesticides. The activity spectrum
of Bt is still expanding. Thus, Bt may provide an alternative to many currently
used chemical pesticides (Lambert and Peferoen, 1992). Various methods are
used to identify the novel activities or rank the relative potencies of Bt strains.
These include bioassay, molecular probe using gene specific polymerase chain
reactions (PCRs) and other.biochemica1 or immunological methods.

2.2 Fermentation

The development of an economical fermentation process involves a great deal of


experimentation and is largely empirical and must be specifically designed for a
particular strain of Bt. Thus the production of a new bio-insecticide using a new
Bt strain requires the development of a different fermentation process. This may
only require minor changes in an existing process the or physiological properties
of the new strain may be different enough to require majorchanges in the entire
manufacturingprocess(DulmageandRhodes,1971andKozielet al., 1993).
Althoughstrains of Bt differ, they allsharesomecommonproperties. It is
therefore possible to define some basic requirements for production of Bt. As
production of &endotoxin occurs only during sporulation, optimal sporulation is
essential. Under anaerobic conditions, growth ofBt is slow and sporulation may
beinhibited;goodoxygensupplyisthereforeanessentialrequirement.High
frequency plasmid loss was induced in Bti by cultivating it at 43C (Gonzalez
and Carlton, 1984). Therefore, cultivation at higher temperature is best avoided.
In case of recombinant Bt containing plasmids, typically, temperature during
production is kept at 28C. Bt is not particularly sensitive to pH and optimum
growth occurs betweenpH 6.5 and pH 7.5 (Bernhard and Utz, 1993).
The cost of production is one of the major issues in commercializing
biopesticides products. The most important factors that determine the cost of
productionareraw materialcost andtheproduct yield. Intherecentpast,
variousrawmaterialsofagricultureorigin and industrial residues have been
investigated to replace the expensive ingredients
currently used in the
manufacture of Bt. The cost of these materials vary between 20 to 60% of the
overallcostofproduction.Theserawmaterialsare wheat bran, rice bran,
defatted soybean cake, powder of legunlinus seeds, molasses, corn steep liquor,
12 Chapter 1

fermented cassava, ground andwhole maize, fish meal etc. The raw material
cost can be minimized by selecting substances which are locally available on
yearround basis, haveminimumenvironmental risks, sustain high specific
growth rate, sporulation and toxinyield and have minimum cost (Tyagi, 1997).
Bt can be produced both in semi-solid and in submerged fermentation,
both of which are used commercially. However, the details of the fermentation,
downstream processing and formulation of Bf products are generally kept as
trade secrets.

a)Semi-solidfermentation:Mechalaspatentedaprocess in 1963 for the


large-scale production of Bt toxins by surface cultures on bran-based semi-solid
media. In a typical production with semi-solid media, nutrients are contained in
a coarse porous matrix with a large surface area through clumping induced by
excessive moisture. In large cultures, oxygen transfer, humidity and temperature
are maintained by either placing the media on shallow trays, or keeping them in
aerated drums. Dulmage (1983)has reviewed semi-solid production ofBt.

b) Submergedfermentation:A typical Bf fermentation(Figure 1.4) is started


by inoculating a flask of culture medium with a loopful of bacteria from an agar
plate. The flask is thenincubated at 28C onashaker. At this stage, spores
germinate and the vegetative cells adapt to the culture medium. After 10-16
hours, the cells are in mid-log phase. Theculture is then used to inoculate a seed
fermenter containing the same culture medium. Counting cells microscopically
monitors growth in the fermenter. After 6-8 hours, the culture is in the late log-
phase and is transferred into the larger fermenter. If the transfer is not delayed
too long, the cells will continue to grow exponentially. Growth in the fermenter
is again monitored microscopicallyuntil the sporangia start to lyse.
There are advantages and disadvantages in each type of fermentation.
Productiononsemi-solid media allow cultivation of microorganismswhich
either do not grow at all or fail to produce the desired product in submerged
culture. Semi-solid media are, however difficult to sterilize and to keep sterile
during
production.
Maintainingdefined,
uniform
conditions
during the
productionprocess as welladjustingparameters like pH, is also difficult.
Therefore, the deep liquid fermentation, in whichgrowthofbacteriumtakes
place in liquid medium in large mechanically agitated vessels sparged with air,
is the preferred method of production for current Bt insecticides (Bernhard and
Utz, 1993).
Bacillus thuringiensis 13

Proteln Carbohydrate
Sterlizer

7 Coo er

I
'A
300 ml
flask
'il-
6 litre
flask

'Seed train'
gj- ,
I
,z7:
gal tank

Filtered
air
:* Screen Ing

Standardizason

Formulation

Packaglng

Figure 1.4 Submergedfermentation of B. thunngiensis

2.3 Formulation

Bt bioinsecticides have been formulatedin many ways since their introduction in


1938. Wettablepowders, dispersible granules, dust, microgranules,aqueous
flowable liquids and oil based flowable liquids that are dispersible in water have
been developed (See chapter4). Early formulations were oftendifficult to apply
and their performance was often poor or unreliable. Advances in application
equipment and improvement in formulation technology have largely overcome
application difficulties. However, different strains maywellrequire different
formulation because of difference in their respective fermentation media and
their by products. Also, batch to batch formulation problems can be encountered
due to variablequality of the fermentation components.

3 Classification and Nomenclature of Bt


As Bt becameincreasinglycommerciallyimportantandnumerous different
isolates werediscovered, the need for amethod to identify and classify Bt
14 Chapter 1

subspecies becomes apparent. They can be classified by a number of techniques


includingserotyping,crystalserology,crystalmorphology,proteinprofiles,
peptide mapping, DNA probing and insecticidal activity.

3.1 Serotypitg of Bt Strain

Vegetative cells of Bt have (at least) two major antigens on their surfaces: the
flagella (H) antigen and the heat-stable somatic (0)antigen (HSSA) (Mike etal.,
1990).The H-serotyping
has been
succesful in theclassification and
identification of Bt strain(deBarjac,1981 and deBarjac & Francon,1990).
More than 40 Bt serotypes are recognised on the basis of flagellar H-antigens.
Sincethe introduction of this method,arapidlygrowinggroup of
serotypes, called serovars have been described. The method is now widely used
by researchers and officials to designate and distinguish individual Bt strains.
Although many researchersuse the word 'variety' for these taxa, according to the
international codeof Nomenclature of Bacteria, the correct name is'subspecies',.
Examplesare B.
thuringiensis subsp. tenebrionis,B.thuringiensis subsp.
aizawai etc.
Despite the fact that the biologicalsignificance of theserotype is
unclear, the method is still used for general classification of Bt. However, the
serotype has certainly very little to do with the insecticidal activity or presence
of certain crystal proteins. For example, Bt subsp. tenebrionis shares the same
antigens as serovar (=variety)morrisoni (8a8b) and can only be distinguished in
terms of theirpathogenicity to coleopteran larvae (deBarjacandFrancon,
1990). There is no immunological relationship between the crystal toxin of Bt
subsp. tenebrionis and &endotoxin of theotherstrainsinH-serotype8a8b.
Further more, Bt subsp. tenebrionis belongsto 0-serotype IX (incontrast to
strainPG-14)(Kriegetal.,1987).Thus,subsp. tenebrionis, which is very
unique in host range crystal morphology, crystal toxic gene and crystal protein
chemistry, does not merit separate status. Moreover, several strains producing
the same crystal proteins with samespectrum of activitybelongtodifferent
serotype. Similary, not all serover aizawai strains are active against the cotton
leaf worm (Spodoptera littoralis), The morrisoni serotype harbors strains that
areactiveagainstLepidoptera (strain HD-12),Diptera(strainPG-14) and
Coleoptera (Bt subsp. tenebrionis). The recent techniques that have been applied
to the identificatiodclassification of Bt are high
performance liquid
chromatography(HPLC),plasmidmapping and cloning and sequencing of
crystal toxin genes.
Bacillus thuringiensis 15

Figure 1.5 Bt crystal inclusions include bipyamidal, sphencal and


rectangular shapes (Source: Lopez-Meza et al., 1995)

3.2 Morphology of Parasporal Inclusions

In general,the shape of the parasporal body is a good but not absolute indication
of anisolate'spathotype. Bt isolateproducewater-insoluble,parasporal
inclusions of a wide range of morphological types. Crystal inclusion can be
classified into various types, such as bipyramidal (Cryl), cuboidal (Cry2), flat
rectangular (Cry 3A), irregular(Cry3B),spherical (Cry4A andCry4B)and
rhomboidal (Cryl 1A) (Figure 1.5).
Bipyramidal crystals show agreater frequency of toxicity than all other
types, and themajority of isolateswithlepidopteranactivitycontain such
inclusions. Irregular spherical crystals can be mosquitocidal, rhomboid are often
active against certain coleopteran species and irregular pointed (eg.triangular)
crystalsincludethosewithnoidentifiedtoxicologicalactivity.Bipyramidal
crystals synthesized in host cells are typically about 1 . 1 long ~ and 0.5pm
wide. Bt subsp. isruelensis (H-14) and Bt subsp. morrisoni (PG-14 isolate),
16 Chapter 1

produce proteins that crystallize in different shapes such as spherical parasporal


body (0.7-lpm) that is toxic primarily to nematocerous dipterans, e.g. mosquito
and blackfly (Federici et al., 1990). Bt subsp. tenebrionis produces a parasporal
crystal that is toxic to certain species of Coleoptera. The crystal appears as flat
plates with square, rectangular or rhomboidal outlines, their axes measures 0.7-
2.4x 0.7pm and the thckness of the plates rangesfrom0.15-0.25pmwith
pointed or roofshapededges(Keller & Langerbruch,1983).Thedegreeof
complexitywithin the parasporalbodiescanbecomposedof single typeof
protein molecule or a mixture of as many as three (Lopez-Meza and Ibarra,
1996).

4 Bt Crystal Proteins and Genes


Crystalproteinshavebeen classified on the basis of their insecticidal and
molecular properties and are assignedcapitalized three letter Roman Code, Cry.
The corresponding geneis assigned three letter lower case italicized code cly.

4.1 Classifcation and Nomenclature

Based on the primary target insect specificity andsequence similarity, the


insecticidal crystal proteins were classified by Hofte and Whiteley (1989) into
five major classes; four Cry classes, Cry (-1, -11,-111 and -1V) and a Cyt class
(Cyt for cytolytic crystal protein). The roman numeral indicated pathotype(I for
toxicity to lepidopterans, I1 forbothlepidopteransand dipterans, I11 for
coleopterans and IV for dipterans in the suborder Nematocera). Feitelson et al.
(1992)added two newmajor classes of nematode-active toxins, CryVand
CryVI to the Hofte and Whiteley classification. The CryV name was given to
protein toxic tobothlepidopteransandcoleopterans.The crystal protein
belonging to eachof these classes were grouped in subclasses (A.B.C....and a, b,
c.....) according to sequence. The upper case letter indicating the chronological
order in which genes with significant differences were described, the lower case
letter in parentheses,indicatedminordifferences in thenucleotidesequence
within a gene type. Thus, CryIA refers to protein toxic to lepidopterous insects
for which the first gene (crylA) was sequenced, whereas CryIVD refers to the
protein for which the encoding gene was fourth fiom this pathotype.
Crickmore et al. (1995) introduced a nomenclature for classifying the
cry genes and their protein products on the basis of comparative amino acid
sequence identity of the full-length gene products. Many cry genes retain the
name assigned by Hofte and Whiteley with a substiution of Arabic for Roman
numerials (eg. crylA(a) become clylAa (Table 1.1 and 1.2).
Bacillus thuringiensis 17

Table 1.1 Homology of amino acids of cry genes

! Honlology of amino
%
Rarrk Symbol gene
acid of cry

Primary Arabic no. 45%

Secondary Upper case letter 75%

Tertiary Lower
case letter wlthout
parentheses 95%

Quaternary Allele number 95%

Table 1.2 Nomenclature of Et Insecticidal protoxins(Adapted


from Crickmore et al., (1 999)

Imecicidal proto.xins
Molecular
Hofte and Whiteley Crickmore et al. Mass (kDa) Tomcity
( 1989) (1 999)

CrylA(a) Cry1 Aa 133.2 Lepidoptera


CryIA(b) Cry1 Ab 131.0 LepldopterdDiptera
CryIA(c) Cry1 Ac 133.3 Lepldoptera
CryIA(d) Cry1 Ad
CrylA(c) Cry1 Ae
CrylB Cry1 Ba 138.0 Lepidoptera
CryIC(a) Cry1 Ca 134.8 LepidopterdDiptera
CrylC(b) Cry1 Cb 134.0 Lepidoptera
CryID Cry1 Da 132.5 Lepidoptera
CryIE Cry 1Ea 132.0 Lepidoptera
CrylE(b) Cry1 Eb
CryIF Cry1 Fa 133.6 Lepldoptera
CryIG Cry9Aa 130.0 Lepidoptera
CrylX Cry9Ba 81 .O Lepidoptera
CryIH Cry9Ca 129.9 Lepidoptera
CryIIA Cry2Aa 70.0 LepidopterdDiptera
CryIIB Cry2Ab 70.8 Lepidoptera
CrylIC Crv2Ac 71 .O LeDidoDtera
Chapter 1

Table 1.2 (continued)

lnsecicidal protoxins
Molecular Mass
Hofte andWhiteleyCrickmore et al. Toxicity
(kDa)
(1989) (1999)

CryIlIA Cry3Aa 73.1 Coleoptera


CryIlIB Cry3Ba 74.2 Coleoptera
CryIIIB2 Cry3Bb
CryIIIC Cry7Aa 73.0 Coleoptera
CryIIIC(b) Cry7Ab 73.0 Coleoptera
CryIIID Cry3Ca 73.0 Coleoptera
CryIIIE Cry8Aa
CrylIIF Cry8Ca
CryIIIG Cry8Ba
CryIVA Cry4Aa 134.4 Diptera
CryIVB Cry4Ba 127.8 Diptera
CrylVC Cry1OAa 77.8 Diptera
CryIVD Cry1 I Aa 72.4 Diptera
CryV Cry1 la 80.0 LepidopterdColeoptera
CryVB Cry1 2Aa
CryVC Cryl3Aa
CryVD Cryl4Aa
CryVIA CryGAa 54.0 Nematode
CryVIB CryGBa
CytA Cytl Aa 27.4 DipterdCytolytic

CytB Cyt2Aa 29.0 DipterdCytolytic

4.2 Bt cry Genes:Encoding Protoxins

Bt toxin genesare usually plasmidbornbutalsochromosomally,theyare


encodedon the transmissible plasmid of molecularweight of 40-150 MDa
(Kumar et al., 1996). The protoxins encoded by cry genes are processed into the
toxin fragments in the midgut of insect and these in turn are responsible for
insecticidal activity.
Bacillus thuringiensis 19

The cry genes encode insecticidal proteins, either of 130 to 140 kDa or
of ca. 70 kDa. For the 130 to 140 kDa proteins, the toxic segment is localized in
the N-terminal half of the protoxin. The C-terminal part of the ca. 130 kDa
proteins (Cryl, Cry4a, and Cry4B) is not essential for toxicity, but is the most
highlyconserveddomain of these crystal proteins. TheCry2A,Cry3and
Cryl 1A (formerly designated as CryIVD) protoxins are smaller.

4.3 Cry Gene Types

a) cryl gene:Genesof the class cryl (eg. crylA,-B, -C etc.)encode130-


140kilodaltons ( m a ) in molecularmassproteinswhichaccumulate in
bipyramidal crystalline inclusionsformerlytermedP1 crystals during the
sporulation ofBt.
The Lepidoptera specific crystal proteins are the best studied crystal
proteins(Massonetal.,1990). CrylA is the most commontypeof crystal
protein that is found in Cryl producing Bt strain and crylAb gene appears to be
most widely distributed gene amongst Bt subspecies. CrylAa is normally highly
toxic for larvaeof the silkworm, Bombyx mori, whereas CrylAc is virtually
nontoxic. CrylC S-endotoxin is toxic to the larvae of the Spodoptera species of
the order Lepidoptera and is one of very few &endotoxin active against these
polyphagous insects (Smith et al., 1996).

b)cry2Agenes:The cry2A geneencode65kDaproteins (fonnely known as


P2 crystal protein), whichoccur as distinct cuboidalinclusions in strains of
several Bt subspecies kurstaki (HD-1, HD-263, NRD-12 and 14 other strains),
thuringiensis,tolworthi and kenyae. TheCry2Aaprotein is toxic to both
lepidopterananddipteran larvae, whereasCry2Abtoxin is only toxic to
lepidopteran insects.

c) cry3 genes: cry3 genesencode


72
kDa
protein
whichform flat
rhomboidal crystals in Bt subsp. tenebrionis, sandiego etc. The Cry3A protein
displays coleopteran toxicity. For most ICP genes transcription is concomitant
with sporulation, howeverCry3Aproteinappearstobeunique in being
expressed during vegetative growth of Bt cell.

d) Bticry genes: The cry4 class of crystal proteingenes is composedofa


rather heterogenous group of Diptera-specific crystal protein genes. The cry4A,
cry4B,crylOA and c r y l l A genesencodeproteinswithpredictedmolecular
masses of 135, 128, 78 and 72 KDa respectively.
20 Chapter 1

e) cyt genes:The27 kDa proteinencodedby cytlA shows no sequence


homologyto the other crystal proteingenes. It is cytolytic fora variety of
invertebrate and vertebrate cells including mammalian erythrocytes. Cyt toxins
are hemolytic and cytolytic in vitro and are specifically active against dipteran
larvae in vivo. The proteins vary in size from 25 to 28 kDa and are smaller than
Cry
polypeptides.
Three
types
have
been
defined
according
to
immunoreactiving.
CytlA is the characteristic cytolytic toxin of Bt subsp. israelensis and
Bt subsp. morrisoni PG-14. CytlAa hasbeenreportedtobehighly toxic to
cottonwood leaf beetle (Chysonzela scripta) and its mode of action against C.
scripta is similar to that observed in mosquito and blackfly larvae (Federici and
Bauer, 1998). It is possible that Cyt proteins may havean even broader spectrum
of activity against insects and owing to their different mechanism of action in
comparison to Cry proteins, mightbeuseful in managing resistance to Cry
toxins.
A second Cyt2A protein is 2kDa larger than the CytlA protein and has
beenfound in the inclusions of Bt subsp. kyushuensis and Bt subsp.
darmstadiensis 73-E10-2. Cytl A and Cyt2A show 70% functional homology
(39%aminoacidsequence identity) (Cannon,1996andHofteandWhiteley,
1989). The CytC is representative of Bt subsp. fukuskaensis. A new type CytD,
has been proposedfor Bt subsp. jegathesan (Guerchicoff et al., 1997).

4.4 Structure of Bt Crystal Proteins

X-ray crystallographyhasrevealed the three dimensional structure of the 6-


endotoxin Cry3A from Bacillus thuringiensis subsp. tenebrionis (Li et al., 1991)
that is specifically toxic to beetles (Figure 1.6), CrylA (Grochulski et al., 1995)
that is toxic to mothlarvaeandCyt2A(Li,Koniand Ellar, 1996),a
mosquitocidal toxin. Cry3A and CrylAa show about 36% amino acid sequence
identity witheach other. However,Cyt2Ashows less than20%aminoacid
sequence identity with CrylAa andCry3A,andhasa radically different
structure.
Cry3A and CrylAa in contrast to Cyt2A, comprise of three domains;
Domain I, is a helical bundle comprised of a centrally located hydrophobic a-
helix surrounded by six additional amphipathic a-helices, thought to be pore-
forming region. The Domain 11, consists of eleven P-strands and one short a-
helix, which are arranged as three P-sheets in a common Greek-key motif. The
P-sheets form the sides of a triangular structure enclosing a central hydrophobic
core. The site directed mutagenesis experiments reveal that this domain is the
one responsible for receptor binding. The Domain 111 of the Bt toxin consists of
twelve additional P-strands in a jelly-roll configuration. The role of the third
Bacillus thuringiensis 21

Domain 111
Domaln I

Domain II

Figure 1.6 Line drawing of the three dimensionalstructure of Cry3showing


distinct structural domains I, 11 and 111 (Source: Li et al., 1991).

domain seems to be closely related to the receptor recognition specificity but its
precise role is still unclear. The unique features of &endotoxins make them an
interesting model protein for studying the relationship betweenstructure and
function (Powell etal., 1995; Yamamoto and Powell, 1993a and 1993b).

5 Insecticidal Activity
Since the first use of Bt as a biological control agent for insects, a great deal of
progress has been made. New Bt strains with improved insecticidal activity have
been identified. New strains with new activities havealsobeen identified,
thereby increasing the spectrum of activity of microbial sprays as a whole.
The host range of Bt has expanded considerably in recent years due to
extensive screening programs, from species in three insect orders (Lepidoptera,
Diptera, and Coleoptera) to species in eight insect orders
(Homoptera,
Orthoptera, Mallophaga, Hymenoptera, Siphanoptera) (Feitelson et al., 1992 and
22 Chapter 1

Feitelson, 1993). In addition groups susceptible to Bt include certain protozoa,


platyhelminths, nematodes, lice, aphids, mites, mollusks and flukes.
The insecticidal activity of a Bt strain is determined by (a) the number
ofcrygenes present, (b) qualitative differences in aminoacidsequences
betweenICPs,(c)difference in expression levels of the genesand(d) the
intrinsic propertiesof the ICPssuch as fermentation stability and activity
(Cannon, 1996).

5.1 Variation in InsecticidalActivityandSpecificity

Thecausesof variation in the insecticidal activity ofICPsincludeboth the


nature of the toxin and the intrinsic susceptibility of the target. The Bt strains
differ not only in their activity towards specific insect species but also in the
relative potency of their 8-endotoxins towards susceptible species. At least three
factors influence the relative potency (or specificity) of ICPs: (a) the origin (i.e.
the Bt strain) of the toxin, (b) the degree of solubility of the protoxin crystals in
the gut juices of the host and (c) the intrinsic susceptibility of the host to the
toxin (Jaquet et al., 1987). Thus the variability of the ICPs themselves, their
solubility and the heterogeneityandconcentrationofreceptors in the target
organism contributeto the specificity of the larvicidal activity.

5.2 Determination of InsecticidalActivity

TOdetermine the insecticidal activity of the crystal protein, the protein is fed to
insects either mixed with an artificial diet or applied onthe surface of leaf disks.
Insect mortality is normallyread after anincubationperiodofseveraldays.
Alternatively, feeding inhibition may be used as an indicator of the activity.
These assays are examples of in vivo, bioassay methods. The crystal protein can
also be assayed in vitro. In this case, crystal protein need to be activated by
incubationwithanappropriate proteinase. Theactivatedtoxincanthenbe
assayedby either an insect cell lysis, toxinreceptor-bindingorpatchclamp
method (Yamamoto and Powell, 1993a).
Bioassay of a microbial insecticide and that of a chemical insecticide
are conceptually different. In the case of a chemical, one determinesthe quantity
and purity (quality) of the active ingredient and uses these data to judge the
insecticidal activity of the product. On the other hand, microbial insecticides
have no tangible active product to be measured. The level of impurities in the
final product is very high; in fact, the active ingredient may represent only a
small percentage of the final product. Furthermore, the level of impurities can
vary widely from fermentationto fermentation.
Bacillus 23

In the case of bioinsecticides, one must measure the insecticidal activity


of the product through bioassays and use this measurement to judge the quantity
and quality of the product. Bioassays are not accessories to the production of
bioinsecticides, they are central and vital to it and the monitoring of entire
production process and the product itself is dependent upon these bioassays.

5.2. I Bioassay of MicrobialInsecticides

Bioassay requires determining the LC50 of the product against test insects. LC5os
by themselves can be misleading since the susceptibility of insect population to
a particular microbial insecticide can vary from day to day.Therefore, it is
preferable to compare the LCso of each test sample with that of astandard
formulation tested at the same time on the same population of insects.For
convenience, a potency value is assigned to the standard in International Units
(IUS) and the activity of the test sample is then also expressed in IUS compared
to the standard.

Calculation of potency in International Units (I.U.) -

LCs0standard x Potencyof standard


Potency of Sample, IU/mg = --
LC50 sample

The activity determined in vivo is greatly influenced by several factors,


including diet, incubation time, incubation temperature, insect age, and insect
behavior. Since the crystal protein is a stomach poison, a sufficient amount of
the protein must be ingested to kill the insect. If the insect rejects the diet
containing the crystal protein, the activity appears low. The rate of crystal
solubilization may be another important factor that can influence activity. In
addition, natural variation (a numerical difference in response that is detected
each time a bioassay is repeated with one genetic strain, either within a single
generation or >1 generation) in response during studies carried out to estimate
potency of microbial pesticides, can lead to erroneous conclusions (Robertson et
al., 1995). For these reasons, it is almost impossible to compare bioassay data
reported from different researchers who use different bioassay methods.

5.2.2Role of Spores in the Toxicity

The role of the spore in the toxicity of the &endotoxin has been subject to
intense debate. Early studies on the insecticidal properties of Bt focussed on the
spore, which at the time was more readily quantifiable than the&endotoxin
components of fermentation products. The spore coat consists of proteins
24 Chapter 1

inlmunologically homologous with those of the crystal. Some insects, such as


wax moth (Galleriamellonelka) and the mediterranean flourmoth (Anagasta
Kuehniella) require bothspores and crystals to be presenttocausedeath.
However, there are many species eg. Pierisbrassicae and Choristoneura
furnijierana, where spores play little or no role in mortality. Rossa and Mignone
(1993) reported for Bt subsp. israelensis that agoodspore count did not
necessarily lead to high larvicidal potency. Milne et al. (1990) conclude that
spore does have a role in determining insecticidal activity, particularly in the
case of species generally less susceptible to Bt. In larvae of these species- eg.
gypsy moth (Lyrnatztria dispar), diamondback moth (Plutella xylostella) and the
beet armyworm (Spodoptera exigulz)- after an initial intoxication resulting from
activation of the Cry toxins in the midgut, the spore germinates and produces
enzymes (phospholipases and proteases). These contribute to the lysis of the gut
cells by degrading cell membranes (Federici, 1998). Van Frankenhuyzen (1994)
suggests that the bacterial specicemia makes an importantcontribution to
mortality in C. jimijierana larvae, and can be initiated by a low level of spores in
toxin-challenged larvae.
In terms of the commercialization of Bt formulations, the focus of
attention has largely shifted away from spores, although there are reports that
their presence can increase the toxicity of ICPs against certain species and also
reduce the effect of Bt induced resistance (see Chapter 5 ) .

5.2.3 Spore
Count

It has been stated in the past that there is a direct and quantitative relationship
between the growth of an insect pathogen and its toxicity toward insects, and
therefore a bioassay test is not needed. In the case of Bt, it was suggested that a
simple bacteriological plate count to measure the spores/ml of a fermentation
broth or the spores/gm of a formulation will indicate how much insecticidal
activity is present. Thus, spore counts could offer an attractive alternative to the
bioassay. Unfortunately, spore counts are essentially meaningless in determining
the potency of a microbial insecticide, as spore count does not reveal the
quantity of &endotoxin produced by an isolate of Bt. Many attempts to replace
the bioassay with spore counts have been made. These have howeverbeen
unsuccessful.

6 Mode of Action
Bt has a wide range of well-characterized insecticidal crystal proteins, which
express as protoxin during sporulation. The crystalline protoxins are inactive. To
be insecticidal, these protoxin must first be ingested by the insect and
Bacillus 25

proteolytically activated to form active toxin. This happens in the insect midgut,
which is also the target organ for Bt toxin. Severalrecentreviewshave
considered the mechanisms or mode of action of Cry toxin (Gazit and Shai,
1995;Himenoand Ihara, 1995;Thompson et al., 1995;Wolfersberger,1995;
Knowles,1994;KnowlesandDow,1993;YamamotoandPowell,1993band
Gill et al., 1992).

6.1 Mechanism

The mechanism of action of insecticidal crystal protein of Bt is a multistep


process. These include solubilization and processing of proteins, toxin binding,
membrane interaction, poreformation, cell lysis and bacterial septicemiaand
death.

6.1.1 SolubilizationandProcessing of Protoxin

Crystal protein activation has been often referred to as a two step process, the
first step involving liberation of the protoxin or crystal protein by dissolution of
the crystal and the second step, proteolytic digestion of the protoxin yielding the
active toxin. Thus,dissolutionanddigestionprocesshasbeentermed as
activation (Powell et al., 1995).
After ingestion, the crystal is first solubilized due to the extreme pH of
the insect midgut, highly alkaline in Lepidoptera and highlyacidic in Coleoptera
larvae (Prieto-Samsonov et al., 1997). The alkaline-solubilized crystal proteins
(usually soluble only above pH9.5) from most Bt strains are about 130 KDa and
require processing by insect gut proteinase. The action mediated by the alkaline
pH and proteinases of insect midguts, yields 60 to 70 KDa proteinase resistant
toxin fragments. The size of this active fragment varies withBt strain.
The midgut environment can play crucial
a role in specificity as shown
with the activation of &endotoxin from B. thuringiensis subsp. aizawai. When
activated with lepidopteran Pieris brassicae (European cabbage worm) midgut
extract, the toxin kills both P. brassicae and dipteran Aedes aegypti (Yellow
fever mosquito) larvae, however when activated by Ae. aegypti midgut extract,
the isolate is toxic only to thesemosquito larvae. Thus,depending on the
proteolytic enzyme,aprotoxincanbeactivated into either adipteranor
lepidopterantoxin
(Koziel
et
al.,
1993).
Differences in the extent of
solubilization sometimes explain differencesin the degree of toxicity among Cry
proteins (Aronson et al., 1991 and Du et al., 1994). A reduction in solubility is
speculated to be one potential mechanism for insect resistance (McGaughey et
al., 1992).
26 Chapter 1

6.1.2 Toxin Binding

The activated toxins bind to specific proteins called receptors located on the
apical brush border membrane of the columnar cells. Much of the host
specificity of Bt toxins is dependent on toxin structure and presence of toxin
receptor sites in the insect midgut. The Cry toxins bind to proteinaceous
receptors in the cell membrane of the insect midgut epithelia. At least two
groups of membrane receptors, cadherins and aminopeptidases have been
identified. Cytolytic toxins, that have significantly different structure, appear to
bind to membrane lipids (Gill et al., 1999). Binding, while essential is not
sufficient to produce mortal damage and although receptor play an essential role,
post-binding factors are required for successful intoxication by Bt &endotoxins
(Bauer, 1995 and Peyronment et al., 1997).

6.1.3 Membrane
Interaction

Upon binding to these specific receptors, the toxin then inserts into the cell
membrane forming pores thereby disrupting cell function. Various modes were
proposed to explain the role of toxin receptor in pore formation, however, the
actual process at the molecular level is not well understood (Gill et al., 1992 and
Knowles and Dow, 1993).

6.1.4 Pore
Formation

The formation of toxin induced pores in the columnar cell apical membrane
allow rapid fluxes of ions. Knowles and Ellar (1 987)proposed the mechanism of
Colloid-Osmotic lysis, which suggests the formation of small 0.5 -1 nm pores in
the cell membrane of the epithelial cells, resulting in an influx of ions
accompanied by aninflux of water.

6. I . 5 Cell Lysis

As a consequence of pore formation, the cells swell and eventually lyse. The
model proposed by Knowles and Dow (1993) placed emphasis on the cessation
of K+ pump that leads to the swelling of columnar cells and osmotic lysis. The
disruption of gut integrity results in the death. The overall midgut pathology of
Bt toxicity results in a loss of bassal involutions in the columnar cells; swelling
of the apical microvilli vesticulation of the endoplasmic reticulum; loss of
ribosomes; swelling of mitochondria; swelling of the cell and nucleus and
subsequent rupture of nuclear, organelle and plasma membrane (figure 1.7) and
Bacillus thuringiensis 27

Microvilli Nudeus

Columnar cell Goblet cell


Normal After 15 minutes

After 30 minutes After 60-90 minutes

Figure 1.7 A schematicrepresentationofthe ultrastructural changes in themidgut


epithelicalcellsof Bornbyx rnori inducedby Bacillus fhuringiensis 6-
endotoxin. With lapsed time, in columnar cells - midgut epithelial cells
showdisappearance of microvilli and basal infolding,swollennucleus
with transformation of mitochondria Into condensed form and the
endoplasmic reticulum into vacuole-like configuration. Simultaneously, In
gobletcells - high electrondensity of cytoplasmandenlargement of
gobletcavityaccompanled with enlargement of intra-space in basal
infoldings take place (Source: Endo and Nishltsutsuji-Uno, 1980).

finally release of the cell contents into the lumen with sloughing of the cells.
Other signs include increase in the number of size of nuclear pores, separation of
cells from each other and from the basement membrane and nearly complete
destruction of goblet cell (Endo and Nishitsutsuji-Uwo, 1980; Luthy and
Ebersold, 1981; Lakhim-Tsor et al., 1983 and Bravo et al., 1992).
28 Chapter 1

6.I . 6 Bacterial Septicemia and Death

Heimpel and Angus (1959) providedthe first concise account of modeof action
of Bt. They noted that the insect midgut membranes are disrupted by the 6-
endotoxin,allowingan ionic flow into the haemolymph.Deathoccurswhen
lysis ofmidgut cells cause irreparable breakdownof the midgut integrity,
allowing Bt and other bacteria present in the lumen to gain access to the body
cavity. The insect haemolymphprovidesanexcellentmediumfor bacterial
growth.Deathcausedbybacteriasepticemiausuallyoccurswithin2-3days
post-ingestion (Knowles, 1994).

6.2 Models for Mechanism of Pore Formation

There are various models proposed for mechanism of pore formation by Bt 6-


endotoxin. Among the proposed models,the umbrella and penknife models,
based on the Cry3A structure, have been reviewed by Knowles (1994). Both of
these models are described below.

6.2.1 Umbrella
Model

As described earlier, the Cry toxin structure consists of three protein domains
each with a specific function. The domain I consist of seven a-helices and is
thought to be involved with membraneinteractions and the insertion of the toxin
into the insects midgut epithelium and pore formation. The domain I1 appears as
a triangular column of three P-sheets and is reported to be involved in receptor
binding.Domain I11 consists ofaP-sandwichand is implicated in insect
specificity and stability of the protein structure and might also participate in
receptor binding (De Maggdet al., 1996).
The Umbrella Model for the mechanism of action of Bt &endotoxin
(figure 1.8A) proposes that the first event in toxicity is the binding of domain I1
of the active toxin to an insect gut receptor. Thisbinding triggers a
conformational change of the protein, opening the toxin Umbrella and causing
it to insert into the membrane forming deltaprotein lined pores that lead to cell
lysis and the eventual death of the insect. In this model, helices a-4 and a-5
drop down into the membrane as a helical hairpin while the rest of the domain I
flattens out. One possible orientation of the toxin as it binds to surface, is in an
umbrella-like molten globule state (Figure 1.8B). It has been suggested that key
feature of this model is that domain I helix 12-5 might be the trans-membrane
segment that formed the lining (Li et al., 1991).
Bacillus thuringiensis 29

One possible orientation


of the toxin as itbinds to
its receptor.

A configurationalchange
triggered by bindingcould
be transmitted from
domain II to domain I via

I" a7 initiating membrane


insertion of two or more
Membrane
\ \ helices.

The Umbrella model:

Helices a4 and a5 drop


down into the plasma
membrane as a helical
hairpin, and the other two
helices flatten out on the
membrane surface,their
hydrophobic faces
towards the membrane

The Penknife model:

Helices a5 and a6 flip in


to the membrane as a
helical hairpin.

Figure 1.8 Models o f the Bt crystal toxm forming a pore through the insect cell
membrane (Adapted from Knowles, 1994).
Chapter 1

6.2.2 Penknife Model

Hodgman and Ellar (1990) proposed helices a-5 and a-6 as the pairs most likely
to form the pore. Helices a-5 and a - 6 are joined at the end of domain I predicted
to be farthest away from the membrane and would therefore have to flip out of
domain I like a penknife fashion and insert into the membrane. The remainder of
the molecule would remain at the membrane surface oron the receptor
(Figure1.K). This model doesnot require rearrangement of the rest of domain I,
although a-4 would probably have to slide downwards relative to a-3. The
authors proposed a formation of hexameric toxin pore (internal pore radius 0.6
nm) lined by six helicalhairpins, each donated by a toxin molecule.
Further studies are needed to provide conclusive proof for these or any
other models that may be proposed to explain the mechanism of action of Et
toxins (Dean et al., 1996; Nunez-Valdez, 1997; Yamomoto and Powell 1993a
and 1993b). Recently, a bivalent two-step binding modelhasbeenproposed
which alternatively may represent a conformational change occurring. Based on
gypsy moth receptor binding studies with a three-domain lepidopterin-specific
toxin CrylAc, the model proposes a receptor recognition and binding to domain
I11 followed by a slower but tighter secondary binding to domain 11. Domain I
potentially inserts upon binding to a receptor in a membrane environment
(Jenkins et al., 2000).

7 Persistence
Et has had considerable use in field and forest to control lepidopterous pests, but
it rarely persists for more than a month to give any degree of long term control
(Pruett et al., 1980). Et spores can survive for several years after spray
application (Addison, 1993), although rapid declines in population and toxicity
have been noted.
The persistence of Et preparations varies markedly according to habitat
type. Density of Bts in granaries is extremely high and probably correlates with
the high density of insects, the stability of the climatological conditions, and the
absence of sunlight (UV radiation), which break downICPs.Only in such
contained environments Et spores and crystals are preserved for sufficient time
to exceed the threshold dose needed for infection, killing larvae, and completing
its growth cycle. The presence of Et in a soil does not indicate any enhanced
value in insect control. Et rarely, if even can initiate an epizootic unless abetted
by external conditions such as the crowding commonly present in insect rearing
facilities (Lambert & Peferoen, 1992; Pruett et al.,1980 and Delucca et al.,
1981).
Bacillus thuringiensis 31

7.1 FactorsAffectingPersistence

The persistence of the introduced toxins is the function primarly of (a) the
concentration added, (b) the rate of consumption andinactivation by insect
larvae and (c) the rate of degradation by the microbiota (Tapp andStotzky,
1995).
Two major technical problems are associated with the effective field
use of microbials, their proper application i.e. their placement where and when
they will exert the most control and their persistence i.e. keeping them active as
long as the destructive stage of the pest population is present.
The loss of persistence of biological activity is the result of multiple
environmental factors such as temperature, water and sunlight.
The
susceptibility of Bt to bio-degradation and inactivation under field conditions
prevents its greater commercial uptake.

7. I . I Temperature

In most agro-ecosystems ambient temperature during the growing season ranges


from about 10 to 40C; however, the optimum range for infection, growth and
development for most entomopathogens lies between 10 and 30" C. In general,
temperature with range of 10 to 30" C for less than 30 days (the period within
which most crops would be vulnerable) does not effect the stability of many
entomopathogens. However, deleterious effects can occur at temperatures less
than 10C or greater than 30" C, when entomopathogensarestressed by
interaction with water, sunlight, foliage or soil chemicals etc.Temperature
above 35" C generally inhibit growth and development of Bt. Insecticidal
activity of Bt is markedly decreased as temperature approach 50C (Table 1.3)
(Ignoffo, 1992).

7. I .2 Water

Water, other than as a dispersal and diluting vehicle or in combinations with


other environmental factors, may limit persistence and subsequent field
effectiveness. The half life of Bt exposed at 50C is greater than 100 days while
wet spores have half-life of less than 60 days. The spores of Bt survive longer if
they are dry. Estimated loss in stability (% in days) at 30C for wet Bt spores is
18% while there is no loss in stability when it is dry (Ignoffo, 1992).
32 Chapter 1

Table 1.3 Bt stability in days at varioustemperatures(Adoptedfrom


Ignoffo, 1992)

Inoculum 5-10C 20-30C 4550C

Spores >5,000 2300 100


Toxin >5,000 90 >90

7.1.3 pH

Soil pH may be an importantvariableaffectingthesurvivalof Bt insoil.


Enumeration ofBt spores on nutrient agarsof different pH showed that optimum
growth occurred at pH 6.7 and 6.4. A 10-fold reduction in numbers occurred
at6.0 and 5.6, a further 10,000-fold reduction atpH 5.1 and no growth atpH 4.4
(Saleh et al., 1970).

7.I .4 SolarRadiation

Natural sunlight (the active spectrum is between 290 and 400 nm) is the most
destructive environmental factor affecting the persistence of entomopathogens.
Sunlightmaydirectlyorindirectlyinactivateentomopathogens.Thedirect
effectsmaybedeletions,cross-linking,strandbreakage,and/orformation of
labile sites on DNA. Indirect effectsmay be due to generation of highly reactive
radicals (e.g. peroxides, hydroxyls, singlet oxygen) producedby near ultraviolet
radiation (UV), which are
primarily
responsiblefor
reducing the field
persistence. If mechanical loss is excluded, the solar radiation would be major
factor affecting the persistence of Bt on treated leaves (Salama et al., 1983 and
Ignoffo, 1992).
The variability in reports onthe persistence of Bt is probably a result of
differences in type of foliage sprayed,the strain of Bt tested, weather conditions
after the spray, differences in the relative susceptibility of the insects used in the
bioassays and the extent of sunlight shielding provided by the plant.

8 Safety and Ecotoxicology of Bt


After more than three decades of operational, commercial applications of many
types of Bt formulation on millions of acres of crops, forests, lakes, rivers and
streams there has never been a reported, document incident involving adverse
field effects onman or the environmental following these applications.
Bacillus 33

No unexpected toxicities have been noticed and no serous outbreak of


Bt in insect populations has been documented. Thisis probably because, Bt does
not survive or grow wellin natural habitats, and has a narrow hostspecificity.

8.1 Mammalian
Toxicity

There are concerns about the mammalian pathogenicity of the genus Bacillus
becauseonemember, B.anthralis, is a virulent mammalianpathogen,and
variousspeciesof Bacillus havebeenassociatedwith infections following
traumatic wounds. For these reasons, Bacillus thuringiensis underwent careful
testing during its commercial development, including full chemical safety tests
and infectivity studies. Like their chemical counterparts, entomopathogens must
be evaluated for their safety to both animals and humans, although the tests used
toevaluate their safety differ fromthechemical safety protocols. Microbial
safety tests concentrate onacute toxicity andvertebrate infectivity, while
chemical safety tests focus on acute toxicity, neurotoxicity, and carcinogenicity.
Theocularanddermal irritancy ofboth chemicalandmicrobialagentsare
additional concerns (Siege1 and Shadduck, 1990).
There were no adverse effects on human volunteers, who were also fed
Bt. Human ate 10" spores per day for five days and inhaled lo9 spores with no
ill effects (Burges,1981). Injecting highdosagesof Bt intracranially and
intraoculary in domestic animals and wild type did not causesignificant toxicity
or infectivity. Conventionalroutesofexposuresuch as oral, parenteral,
respiratory and dermal also showed no toxicity or pathogenicity (Saik et al.,
1990).
TheCryproteins typically requireboth solubilization and activation
steps beforetheybecomebiologically active toxins. Formost, solubilization
occurs in thehighly alkaline environmentoflepidopteran insect midguts.
Activation occurs via discrete proteolysis by insect gut enzymes and may occw
concomitantlywith the solubilization step. Thehighly acidic nature of most
mammalian guts is not a favorable environment for the Cry toxin. The low pH
of most mammal guts would solubilize and denature the Cry proteins, making
themsusceptible to hydrolysisbynativegutproteases into inactive small
peptides and free amino acids. As such, all the literature reports conclusively
support the observation that Bt is not a mammalian pathogen.

8.2 Toxicity to Avian Species and Fish

Bt was fed to avian species for as long as 690 days with no adverse affect. Bt
was found to be not toxic to wildlife including birds and fish and non-target
vertebrates or invertebrates in the terrestrial or aquatic habitat (Burges, 1981).
34 Chapter 1

8.3 Affect on Nun-TargetOrganisms(NTOs)

One of theoutstandingadvantages of Bt is safety for insect parasites and


predators. Bt has no apparent effect on beneficial insects such ashoneybees
(Bailey, 1971 and Burges, 198 I). The lepidopteran active varieties of Bf
however, exhibit mild toxic effects on some invertebrate NTOs at recommended
label rates.Many of these effects are secondary in nature, resulting from the
declining health of the host or prey larva (Melin and Cozi, 1990).

8.4 Affect on BeneficialInsects

8.4. I Silkworm (Bombyx mori)

Concern over potential harm to silkworm has led some countries to prohibit the
use of Bt product, a position that mightnow logically be resisted given the
diversity of available Bt strain (Driesche and Bellow, 1996). Bt subsp.
thuringiensis that are effective against pest insects, are relatively harmless to
silkworms. However, what is harmless for silkworms is not necessarily harmless
for other beneficial insects. e.g. Bf subsp. fhzrringiensis is more harmful than Bf
subsp. souo to honeybees, but subsp.sotto is oneof the most harmful to
silkworms (Bailey, 1971).

8.4.2 Honey-bees (Apis mellifera)

Research by several workers failed to reveal Bf activity against adult or


immature honeybees. Beeswerefedwith sugar solution containing Bt subsp.
fhzrringiensis spores (0.67 and I .67 X I O9 per bee), supernatant (2.5 mg per bee),
and crystals (0.5 to 16X IO6 per bee) and also crystal of subsp. alesfi and sotto
(both at 0.5X 1 O6 per bee). All three crystal types failed to harm the bees, but the
p-exotoxin (supernatant)gave nearly 100% mortality at 7th day. Significant
mortality was seen in the spores treatment at 8th day. Thiswas probably a
consequence of septicemia(Cantwell et ai., 1966).

9 Concluding Remarks
Because of their environmental safety, microbial insecticides are one of the few
pesticides that can be developed and registered quickly and cheaply. Pressure for
non-hazardous, environmentally compatible pest-control measures has spurred
Bacillus thuringiensis 35

the interest in Bt,whichisnow widely acknowledgedas an interesting and


promising Source of insecticides. In addition, resistance to conventional
insecticides does not confer cross-resistance to Bt toxins due to the unique mode
ofaction ofs-endotoxin.Bt toxins would contribute to reduction of the chemical
insecticides load, reduced pressure on beneficial insects andother non-target
organisms, and increase worker safety by reducing exposureto pesticides.
Ongoingscreening programswill undoubtedly reveal Bts withnew
activities in terms of increased toxicity and new spectra of activity. The highly
specific toxicity of the ICPs has raised the interest of scientists and industry. As
lCPs can be readily producedby fermentation, the development of relatively
economical biopesticides is practical. The bacterial production of ICPs and its
release as a crystal in a stable, inertform facilitates the production of
commercial sprays.
Themajorimpetusfor greater use of Bt in agriculture is the
development of resistance to conventional insecticides. Today, Bt-based
insecticides are frequently used in intensive agriculture, (a) in conjunction with
conventional insecticides (see Chapter IO) as a backup for control failure, or (b)
last resort once resistance to other insecticides has occurred (Bauer, 1995).
Despite many appealing characteristics, the use of conventional Bt-
based insecticides is often constraineddueto(a) verynarrow specificity. (b)
maximum effectiveness being limited to particular developmental stage of the
pest, (c) short shelf life, (d) low potency, (e) lack of systemic activity and (f) the
presence of viable spores. The specificity is a problem for major world crops
that have pest complexes and a single Bt-based product cannot control them.
The insecticidal microorganisms or their toxic products are sensitive to
environmental factors such as ultraviolet light, plant surface chemical, heat and
desiccation. The Bt &endotoxin is short-lived on crops, necessitating the need
for many applicationsduring a growingseason. Bt, as with other spray-on
pesticides, is difficult to deliver to insect speciesthat burrowinto their host
plant, hide under leaves or live primarily under the soil surface. In caseof
aquatic insect control, maintaining the 6-endotoxin in the water at the level of
insect feedingzone is difficult. Because Bt kills insects slowly and has low
residual activity, it must be used prophylactically, and Bt-based products cannot
control well-established insect populations. The presence of spores in Bt
products is a problem in countries like Japan, because a massive release of
spores would potentially threaten the silk industry, which uses the Bt-sensitive
silkworm.
Finally, potentially although 51 provides an alternative to chemical
insecticides, totally substituting Bt for the use of chemicals would be a mistake.
Use of the chemical arsenal in combination with Bt would probably enable a
more judicioususe of both and would also delay the onset of insect resistance.
36 Chapter 1

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production in fed batch cultures ofBt, Biotecllrlol. Letts., 15,295-300.
Bacillus thuringiensis 41

78. Rowe, G. E.and A. Margaritis,1987.Bioprocessdevelopments in the


production of bioinsecticides by Bacillus thuringiensis, Crit. Rev. Biotechnol.,
6, 87-1 27.
79. Saik, J. E., L. A. Lacey and C. M.Lacey,1990.Safetyofmicrobial
insecticides to vertebrates - DomesticanimalsandWildlife, in Safety of
MicrobialInsecticides, L. Marshall, L. A. Laceyand E. W. Davidson,eds.,
CRC Press, Florida.
80. Salama, H., M. S. Foda, F. N. Zaki and A. Khalafallah, 1983. Persistence of
Bacillus thuringiensls Berlinerspores in cottoncultivations, Z. Angew.
Entornol., 95, 321-326.
81. Saleh, S. M., R. F. Harris and 0.N. Allen, 1970. Fate of Bacillus fhurlngiensis
in soil: Effect of soil pH and organic amendment, Can .J. Microbiol., 16, 677-
680.
82. . . E.,. N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J, Feitelson, D. R.
SchneDf.
Zeigler and D. H. Dean, 1998. Bacillus thuringiensis and its pesticidal crystal
proteins, Microbiol. Molecular Biol. Revs., 775-806.
83. Siegel, J. P. and J. A. Shadduck,1990.Mammaliansafetyof Bacillus
thuringiensis isruelensis. in Bacterial Control of Mosquitoes and Blackjlies,
H. de Barjac and D. J. Sutherland, eds., Unwin Hyman, London, pp. 202-217.
84. Smith, G. P., J. D. Merrick, E. J. Bone and D. J. Ellar, 1996. Mosquitocidal
activity of the CryIC 6-endotoxin from Bacillus thuringiensis aizawai, Appl.
Environ. Microbiol., 62(2), 680-684.
85. Sonngay, S. and W. Panbangred, 1997. As reported in Schnepf et al., 1998.
86. Stabb, E. V., L. M. Jackson and J. Handelsman,1994.Zwittermicin A-
producingstrainof Bacillus cereus fromdiversesoils, Appl. Environ.
Microbiol., 60,4404-441 2.
87. Steinhaus, E. A,, 1951. Possible use of Bacillus thuringiensis as an aid in the
biological control of the alfaalfa caterpillar,Hilgurdiea, 20, 359-381.
88. Tapp, H. andG.Stotzky,1995.DotblotELlSAformonitoringthefateof
insecticidal
toxinsfrom Bacillus thuringiensis in soil, Appl. Environ.
Microbiol., 61(2), 602-609.
89. Thompson, M. A,, H. E. Schnepf, andJ. S. Feitelson, 1995. Structure, function
andengineeringof Bacillus thuringiensis toxins, in Genetic Engineering:
Principles and Methods, Vol. 17, J. K. Setlow, ed., Plenum Press, New York,
NY, pp. 99-1 17.
90. Tyagi, R. D., 1997.Rawmaterialsfortheproduction of biopesticides in
developing countries, TCDC Intl. Workshop on Application of Biotechnology
in Biofertilizers and Biopesticides, Oct. 15- 18, 1997,IITDelhi,Abstracts,
pp.89-92.
91. van Frankenhuyzen, K., 1994.Effect of temperatureandexposuretimeon
toxicity of Bacillus thuringiensis Berliner spray deposits to spruce budworm,
Choristorleuralum~eranaclemens (Lepidoptera: Tortricidae), Can. Entonlol.,
122,69-75.
92. Villafana-Rojas, J., E. Gutierrez and M. de la Torre, 1996. Primary separation
of the entomopathogenic products of Bacillus thuringiensis, Biotechnol. Prog.,
12,564-566.
42 Chapter 1

93. Wolfcrsberger, M. G., 1995.Permeabilityof Bacillus thurirzgiertsis Cry I toxin


channels, in Molecular Action of Insecticides on Ion Channels, J. M. Clark,
ed., American Chemical Soclety, Washington, DC.
94. Yamamoto, T. and G. K. Powell, 1993a.
Structure and function of the
Insecticidal protein produced by Bacillus thuringiensis, in Recent Adv. Mol.
Biochem.Res.Proteins, Proc.,IUBMBSymp.Struct.Funct.1992 (Publ. 93).
Yau-huei Wei, Ching-San Chen and Jong-Ching , eds., World Sci., Singapore,
pp. 137-144.
95.
Yamamoto, T. and G. K. Powell,
1993b. Bacil/zcs thztringlensis crystal
proteins:
Recentadvances in understanding its insecticidalactivity, In
Advanced Engineered Pesticides, Leo Kim, ed., Marcel Dekker, Inc.,New
York. NY, pp. 3-42.
Bacterial Insecticides for
Crop and Forest Protection
and Insect Vector Control

1 Introduction
Microorganism based biopesticides have attracted greater attention during last
decades for biological control of plant pests. The use of microorganisms as a
source of biological compounds for insect pest control startedwith the discovery
of the highly insecticidalbacteria Bacillusthuringiensis. Byfar,themost
successful of microbial insecticidein agricultural and forest insect pest controlis
Bacillus thuringiensis subsp. kurstaki (Btk). Until the mid 1970s, Bt (mainly Btk)
was thoughtonly to produceproteins that were insecticidal to lepidopterous
insects. In 1976, Goldberg and Margalit isolated a new subspecies of Bt named
israelensis (Bti) whichwas found to be a pathogen of mosquito larvae. This
discovery of anew subspecies of Bt led to much research into its possible use of
mosquitocontrol. After the successfuldevelopmentof Bti asalarvicidefor
somemosquitoesandblackflies,another Bacillus species, sphaericus was
developed to complement Bti in the controlofvariousmosquitospecies in
diverse habitats. Detailed descriptions of individual characteristics, insecticidal
properties and safety profiles of all the three above mentioned Bacillus species
are provided in this chapter.

43
44 Chapter 2

2 Bacillusthuringiensis subsp. kurstaki


Btk was first isolated in 1962 by Edourad Kurstak from diseased Mediterrean
flour moth (Anagasta kuekniella) larvae from a flour mill at Bures Sur Yvette
near Paris, France. Abbott Labs introduced the first commercial Product 'DiPel'
in 1970. Since then, many isolates of Btk eg. HD-1 isolated from diseased pink
bollworm (Pectinophora gossypiella) by Dulmage (1970) and HD-263 isolated
from dead almond moth (Ephestia cautelln) by Ayerst proved to be superior
against allHeliothis spp. tested.
Btk specificity for Lepidoptera and short persistence in the environment
makes it an attractive alternative to synthetic chemical insecticides in many
agricultural and forest ecosystems. It is widely used against lepidopteranpests in
cotton, cornandsoybeancropsand is effective against foliage feeding
caterpillars. It has also been successful in controlling forest pests infestations
such as spruce budworm and gypsymoth (Spear, 1987).
Thereare also someotherlepidopteran-active Bt subspecies.These
include aizawai HD-I 12 and HD-133 and morrzsoni HD-12. Also, there is a
coleopteran-specific Bt subsp. tenebrionis (sandiego), which is effective against
several beetlespecies
such as, Colorado potato
beetle (Leptinotarsa
decemlineta) Alder leaf beetle (Agelastica h i ) , Cottonwood leaf beetle
(Chtysomelascripta), Eucalyptus tortoise beetle (Paropsischatybdis) etc.
(Keller and Langenbruch, 1993).

2. I Characteristics

Btk is widely distributed, rod shaped, aerobic, gram-positive,


crystalliferous, spore forming, soil bacteria belonging to the family Bacillaceae.
Btk is givenaserotypename3a3b. It is differentiated fromother Bts by
comparing their serotypes.
During sporulation, in addition to endospore, it produces a parasporal
body that contains one or more proteins typically in a crystalline form. These
crystalline proteins known as &endotoxinsoccur as protoxins,whichupon
activation exhibit ahighly specific insecticidal activity againstlepidopterous
larvae.

2.2 Btk CrystalProteinsandGenes

2.2.1 Parasporal
crystal

Most isolates of Btk (e.g. HD-73) produce a large single bipyramidal parasporal
crystal (1.1 x 0.5pm) containing single protein that is almost always only toxic
Bacterial 45

to lepidopterousinsects.However in someisolates of Btk (e.g. HD-l), the


bipyramidal crystal is accompanied by smaller cuboidal crystal and are toxic to
lepidopterans and mosquitoes (Figure 2.1) (Federici, 1993; Cannon, 1996).
The bipyramidal protein crystal has a composition as follows: CrylAa,
13.6%, CrylAb, 54.2% and CrylAc, 32.2% (Masson et al., 1990). On the other
hand, the cuboidal crystal inclusionconsistsofCry2Aproteins,which is
responsibleforweakbutsignificanttoxicity to mosquitolarvae in some Btk
strains such as HD-1, HD-263and NRD-12. The kurstuki strain HD-263 showed
superioractivitywhencompared to HD-1againstseveralmajoragricultural
pests although the insecticidal spectrum of the latter is broader. HD-73 is more
activethanHD-1against Heliothis spp. and alsoCodlingmoth (Cydiu
pornonella) (Navon, 1993).
Btk HD-1 protoxin genes show 99% homology with Bt subsp. sotto,
91%with berliner and85% with kustnki HD-73.Thebipyramidalprotein
crystals of both Btk strains, NRD-12 and HD-1, contain all the three c l y f A gene
products ( c l y f A a .-b and -c); however only the CrylAa and CrylAb component
vary between crystals (Massonet al., 1990).

2.2.2 Btk encodinggenes

c l y l A gene appears to be the most widely distributed gene amongst differentBtk


strains and encode 130-160 kDa protoxins. These Lepidoptera specific proteins
are converted by proteolysis into a toxic core fragment of 60 to 70 kDa (Hofte
and Whiteley, 1989). Similarly, the genes of the class cry2 that encode 65 kDa
proteins also maintain Lepidoptera activity and occur in several Btk strains -
such as HD 1, HD-263 and NRD-12. cry2A gene product is active against both

Figure 2.1 Electronmicrograph of Bacillus tkwinglensis subsp. kurstakr strain HD-I


showing crystal and spore (Spear,1987).
46 Chapter 2

Lepidoptera and Diptera species. Of the five protoxin genes occurring in Btk
HD-1, four of its cry genes ( c ~ y l A acryIAc,
, cry2Aa and cry2Bb) are carried on
a single 110-MDa plasmid (Carlton and Gonzalez, 1985) and the remaining cry
gene (crylAb) occurs on an unstable, smaller 44-MDa plasmid. The c ~ gene y
from Btk strain HD-1 was first cloned by recombinant DNA techniques in 1981.
Sincethen many protoxin genes havebeenclonedandsequenced(Cannon,
1996).

2.3 Insecticidal
Activity

Btk acts specifically against many species of Lepidoptera, with the most notable
being the cabbage looper, tobacco hornworm, tobacco budworm, Europeon corn
borer,gypsymothandsprucebudworm.Usesforwhich Btk is an accepted
insecticide rangewidelyandinclude forestry, vegetables,corn,tobacco,
ornamentals, fruit trees andstored grains. The success of Btk is based on a
combination of efficacyandsafety.Several pests ofagronomicimportance
controlled by Btk and the related target crops are givenin Table 2.1.

Table 2.1 Commonpestscontrolledby Bfk andtheirtargetcrops

Pests Target crops

Anticarsia genlnrafalis
Soybean, sunflower and peanuts.
(Velvetbean caterpillar)
Argyrofaettia spp. Pomefruits, currants (blueberries, caneberries, black-
(Tortrlx moth) berries, dewberries, raspberries, strawberries), citrus
(orange, lemon, grapefruits, tangerine, pamclo).
Choristot~errraspp. Peanuts, soybean, forestry and shade trees.
(Spruce budworm)
Esfigt~lerre acrea Tomato, peppers, eggplant, safflower, sugarbeet, mints,
(Saltmarsh caterpillar) grapes, small fruits, cane and bush berries, soybean and
other legume crops.
Helicoverpa/ Heliothisspp. Tobacco, tomato, pepper, eggplant, corn/ maize, cotton,
(Budwomdfruitworm flowers and ornamentals.
bollworms )
Hypharrtria spp. Stone fruits,nut trccs, pomegranates, forestry, shade trees,
(Fall webworm) sugar maple trees ornamentals and flowers.
Lobesia botrar~a Grapes
(Grape moth)
Bacterial 47

Pcsts Target crops

Lyrlrantria dispar Pome fruits, forestry and shade trees, sugar maple trees,
(Gypsy moth) flowers and ornamentals.
Malacosonla spp. Pccan, walnut, pome fruits, stone fruits,(almond, cherries,
(Tent caterpillar) peach, nectarines), forestry and shade trccs.
Marrtestra brassicae Cabbage, cauliflower, brassicas, broccoli, brussels sprout,
(Cabbage moth) collard, kale, mustard, leafy vegetables (cclcry, cicory,
saled), and sugarbccts.
Marlduca spp. Cole crops and vegetables, tobacco, tomato, pepper and
(Hornworm) eggplant, flowcrs and ornamentals
Ostrirlia trubilasis Corn/maize and other cereals.
(European cornborer)
Phologoplrora tnetuculosa Leafy vegetables, (celery, cicory, saled) tomato, pepper,
(Angleshade moth) and cggplant.
Platipherla scabra Pcanuts, soybean, pasture (alfalfa, clover, grasses), cole
(Green cloverworm) crops and vegetables, potatoes, cucurbits and sunflower.
Platinota stultarla Flowers and ornamentals, grapes, small fruits, cane and
(Omnivorous leafroller) bush berries, stone frults,nut trees and pomegranates.
Plutella xylostella Flowcrs and ornamentals, cabbage, cauliflower, brassicas,
(Diamondback moth) broccoli, brussels sprout collard, kale and mustard.
Spodoptera spp. Cotton, sugar beet, tobacco, com/maize, grapes, small
(Armyworms) fruits, berry, hops, alfalfa,,cole crops and vegetablcs,
soybean and other legume crops, cucurbits, tomatoes,
pcppcr and eggplant.
Trichoplusia ni Cabbage, cauliflower, brassicas, broccoli, brussels sprout
(Cabbage looper) collard, kale, mustard, beans,peas, tomato, pepper,
cggplant, cucurbits (melon, water mclon, cantaloupe,
squash, cucumber), sugarbeet, mint, tobacco, cotton and
alfalfa.
Tortrix sp. Flowers and ornamentals, forestry and shade trees.
(Leaffolders)
Udeaaferrygallis Leafy vegetables (celery, cicory, saled), peanuts, soybean,
(Lcaf trier) flowers and ornamentals,

Various lepidopteran-specific commercial products based on different strains of


Bt-kurstaki are given in Table2.2.
48 Chapter 2

Table 2.2 Commercial products based on different strains of Bt subsp. kurstaki

Btk Crystal Proteirl Product


strain Manufacturer
Conruositiorl Trade Narne

Cry1
Aa, CrylCry1
Ab, DiPel
Ac,
*Abbott Labs.
Trilogy
HD-lThermo
Thuricide
Cry2Ab
Cry2Aa,

Cry1 Aa, Cryl Ab, Cry1 Ac,


NRD"2 Cry2A Javelin Thermo Trilogy

HD-263 CrylAb,
CrylAc,
Cry2A BMP-123
Beckcr
Microbial

SA- 1 1 Delfin
Trilogy Thermo
* Since February 2000, Abbott Ag specialities products are owned by Valent
Biosciences Corporation, a subsidiary of Sumitomo Chemical Company.

2.4 Mode of Action

Mode of action of Et has been described in Section 6.0 of Chapter 1. Btk is a


stomach poison and has no contact action. Larvae stop feeding on the treated
plants within short time after the ingestion of a lethal dose of Btk. Resulting
death usually occur within 2 to 3 days without further feeding. In commercial
terms, a larva that stops feeding is no longer considered a pest. Only larvae are
susceptible, whereas eggs or adults are not affected. Surviving larvae are
particularly susceptible to natural controlling forces such as insect viruses, fungi,
parasites, predators and environmental stressfrom weather extremes. Even
larvae surviving to pupation may give rise to pupae below average weight and
adults, which may be small, deformed and sterile. Thespecific mode of action of
Btk on caterpillars is depicted in Figure 2.2. The Btk toxins mainly affect the
anterior zone of the midgut of lepidopteran larvae. The midgut of the
lepidopteran larvae is a simple, tubular epithelium that dominates the internal
architecture of the insect. The tissue is composed of two majorcell types;
columnarcells with a microvilli apical borderanda unique gobletcell,
containing a large vacuolar cavity, linked to the apical surface by an elaborate
and tortuous valve. The K' pump is located in the apical membrane of the goblet
cell, pumping Kf from the cytoplasm into the cavity and thence to the gut lumen
via the valve. This electrogenic K' transport is the predominant feature of the
larval lepidopteran gut. Lepidopteran larva is characterized by a high blood ratio
of K' : Na'. Another important feature of the midgut is that the pH of the
lumenal fluid is about 12 (Knowles, 1994 and Kumar et al., 1996).
Bacterial 49

Bt crystalline toxh

crystalline toxin)

Within minutes, the toxin


2 binds to speafic receptors
in the gut wall and the
caterpillar stops feeding

Within hours, the gut wall


breaks down, allowng spores
and normal gut bacteria to
enter the bodycavity; the toxin
dissolves

In 2-3 days, the caterpillar


4 dies fromsepticemlaas
spores and gut bactena
proliferate in the blood

Figure 2.2 Mode of action of Bacillus thuringrensis subsp. kurstaki on


caterpillars (courtesy:Valent Biosciences Corporation).

2.5 Persistence

On agricultural crops, most of the activity of Btk towards target Lepidoptera


disappears within 3 4 days after application (Beegleet al., 1981). The low field
persistence of this insect pathogen is a major problem regulating its effective use
for pest control.

2.5.1 Persistence in water

Field studies have shown that spores of Btk persist for some time in fresh water.
Inlaboratory,survivalstudiesfor Btk infourdifferenttypesofwater i.e.
distilled water, tap water, lake water and sea water at 20' C have shown that Btk
survived for an extended period in all the four tested media. The survival pattern
of Btk in distilled and tap waters show that approximately50% of the original
cell population died off rather rapidly during the first 20 days period, then
50 Chapter 2

declinedmoresteadily andremained relatively unchangedthroughoutthe


remaining part of the experimental period from40 to 70 days.
There is a significant difference in the survival of the Btk in lake and
sea water. Btk was relatively more persistent in fresh water than in sea water.
The viability of Btk in lakewaterremained quitestableafter 50 days. In
contrast, there was acontinuousdecrease of Btk population in seawater.
Approximately 90% of theBtk population died off after 30 days exposure to sea
water. The prolonged survival of Btk in lake water was postulated to be due to
the presence of higher concentrations of available nutrient which may enhance
the growthofbacteria whereas,seawater is generallyconsidered to be
bactericidal to non-marine bacteria (Menonand De Mestral, 1985).

2.5.2Persistence in soil

Spores of Btk have been reported to persist in soil upto a year or more (Delucca
et al., 1981). Bt spore can remain viable for long periods of time in soil in the
absence of germination-inducing stimuli. Under conditions favoring the growth
of soil bacilli, such as neutral pH conditions and the presence of proteinaceous
amendments, Btk can germinate, compete vegetatively
with
soil
microorganisms, and sporulate successfully to attain levels higher than 1 million
sporedgm soil. Btk does not survive if sporegerminationisinducedunder
conditions unfavorable to the competitive growth of soil bacilli (Saleh et al.,
1970).

2.5.3Persistence on foliage

Persistence of Btk in forest system appears to be more variable. Reported half-


life varies from 1 day on oak (Quercus spp.) and redbud (Cercis canadensis)
towards gypsy moth,to 3.5 day in a mixed coniferous forest with activity toward
westernsprucebudworm (Choristoneuraoccidentalis) onsome branches
persisting 20 days after spraying (Johnson et al., 1995).

2.5.4Factorsaffectingpersistence

The impact of environmental factors (primarily sunlight, temperature, humidity-


water) on the field persistence of Bt has been discussed in Chapter 1, Section
7.1. However, the sunlight which is probably the most destructive environmental
factor affecting theBtk is discussed here in detail.
Bacterial 51

Salama et al. (1983) found that one day of the direct sunlight could
inactivate over 90% of Btk spores on potted white spruce. The trees themselves
in the dark can inactivate 78% of the spores in 14 days. They also reported that
Btk spores had half-lives between 75 and 256 hr on cotton leaves, not due to
high temperatures, but rather due to the effect of ultraviolet radiation. Spore
viability of Btk was reduced 50% after 30 minutes exposure to simulated
sunlight. Endotoxin activity also was reduced, however, it required about 8
times more light exposure (3.8 h) to obtain a 50% loss in insecticidal activity
(Ignoffo, 1992). Wavelengths in the 300-380 nm range of the solar spectrum are
largely responsible for loss of toxicity in purified Btk HD-1 and HD-73 crystals.
Sunlight radiation has been shown to cause tryptophan destruction in protein
crystals of Btk HD-1 and NRD-12 (Pozsgay et al., 1987).

2.6 SafetyandEcotoxicologicalEffects

The safety of Btk is not only beneficial environmentally but also leads to other
practical advantages. Unlike most other insecticides, Btk does not require special
protective clothing, there is no waiting period before re-entering the field, and it
may be applied up to the day of harvest. Furthermore, it can be used for aerial
spraying of residential areas for control of gypsy moth, without fear of harm to
human or pets (Spear, 1987).

2.6. I Mammaliansafelyof Btk

Btk has not demonstrated evidence of toxicity, infectivity, irritation or


hypersensitivity to mammals. Research workers, manufacturing staff or users
have observedno allergic responses or health problems. Human volunteer
ingestion and inhalation of Btk led to no ill effects. No toxicity in mice, rats or
dogs has been demonstrated with single dosage of Btk technical up to 10,000
mgkg of body wt. Thirteen week dietary administration of technical material to
rats at dosagesof 8,400 mg/kg/day produced no toxic effects. Two years chronic
dietary administration of technical material to rats at 8,400 mg/kg/day produced
no tumorigenic or oncogenic effects.
No corneal opacity was observed in rabbits treated with 0.1 ml of B f k
technical. There is also no evidence of sensitization in guinea pigs treated with
repeated subcutaneousinjections of Btk technical material.
52 Chapter 2

2.6.2 Toxicity /pathogenicity to bird and,fislt

Studies show that Btk is not toxic or pathogenic to fish or avian species. Btk,
when fedto rainbow trout (Oncorhynchus mykiss), blue gills, bobwhite quail
and for 14 days to chickens,did not produce toxicityto these species.
Anavianoralpathogenecity and toxicitystudyin the mallardduck
(LDso >200 gms Btkkg) indicateslowtoxicity ofBtk to mallard.Similarly,
another avian oral pathogenecityand toxicity study in the bobwhite quail,(LDSo
>10 gms Btklkg) has shown no adverse effects and autopsy of the bird revealed
no pathology attributableto Btk.
In the laboratory, when Btk was added to water containing the marine
fish (Anguilla anguilla), no observable toxic or pathogenic effects was observed
(Burges, 1981 and Product brochure of DiPel@ by Abbott, 199 1).

2.6.3 Effects on non-target organism (NTOs)

Btk demonstrated little or no observable toxicity to non-target organismsin both


controlled testing and actual field usage. No significanteffect of Btk on
Zooplanktonincluding rotifers, coperods,
cladocerans,
phantommidges
(Chaoborus s p . ) and particularly on Daphnia sp. was found in 3 months study.
Btkhasno toxiceffectsonmicrocrustaceans(Coperoda,Ostracado), mites
(Hydracarnia) and insects (Diptera,Heteroptera,Ephemeroptera,Odonata and
Coleoptera).
Whenexposed to concentration of Btk equivalent to theworst-case
field situation,none of larvae of Trichoptera,Plecoptera,Ephemeroptera,
Megaloptera and Diptera species were found to be susceptible, except Simulium
vittatuln. No toxic effect was observedonmussels (Mytilus edulis);oysters
(Crassostrea gigas and C. virginica); common periwinkle (Littorinn littorea);
freshwater shrimps (Crangon crangort) and the brine shrimp (Artentia salina),
when exposed to Btk in aquaria at concentration of 10 to 400 mg/l for 96 h, brine
shrimp was the onlyspeciesfound to besusceptible to the Btk in this study
(Melin and Cozzi, 1990 and Johnson et al., 1995).

2 . 6 . 3 ~ Effect on parasites

Parasitesbeingimportantregulatorsofinsectpestpopulationshavebeen
extensively tested for sensitivityorsusceptibility to Btk. The following
observations have been made:
Bacterial 53

Btk did not affect the parasitism of tachinid species such as Blepharipn
scutellata and Pnrasitigena agilis and there is a reported case of increase
in parasitism by the tachinids, Compsiluraconcinnata and Blepharipn
pratensis.
When washed spores and crystals of Btk (5x10 spores + crystals per ml)
were fed to adult Trichogrammacncoeciae, no mortality or reduced
capacity to parasitize was observed after 7 days feeding (Hassan et al.,
1983).
No decrease in the percentage of parasitism of aphids by Dinretiella
rapue was found on collards treated with Btk.
On aerial spray of Btk, there was increase in the percentage of parasitism
of gypsy moth larvae by Cotesia melanoscelus and Phobocampe
unicinctu. (Ticehurst et al., 1982; Weseloh et al., 1983, and Webb et al.,
1989).

2.6.33Effectonpredutors

It is a matter of concern if a beneficial insect predator may become intoxicated


or infected when feeding upon a pest species that has ingested Btk spores andor
crystals. The following observations have been made:

When lethal quantities of Btk was fed to larval cabbage looper


(Tricoplusia ni) and just prior to death, these larvae were offered to young
Chinese praying mantids (Tenoderaaridijolia ssp. sinensis), it was
observed that mantids were not susceptible to spore/crystal mixtures in an
intact insect host.
When striped earwig (Labidura riparia) an important insect predator of
lepidopteran larvae is treated with Btk at rates equivalent to 10 times the
normal field application rate, no mortality was observed.
Btk has shown an effective control of lepidopteran pest species with no
detrimental effect on nymphs or adults of spined stiltbugs (Jalysus
spinosus) important predators on lepidopteran eggs, particularly those of
tobacco budworm (Heliothis virescens), during a 2 months long study.
No toxic effect has been observed on the spined soldier bug (Podisus
maculiventris ) following forest spray of Btk on the oak leaf caterpillar
(Heterocampa manteo)(Melin and Cozzi, 1990).

2 . 6 . 3 ~ Effectonbenejicials

No adverse effect of Btk on beneficial arthropods, predators or parasites has


beenobserved during laboratory and field studies.Those studied include
54 Chapter 2

predaceous bugs, big eyed bugs, damsel bugs, assain bugs, lacewings, lady bird
beetles, soft winged flower beetles, parasitic wasps, paper-nest wasps,etc.

i) Effectonhoney bees: Btk has no toxic effects on honeybeesand


when fully sporulated culture of Btk was fed to adult bees at conc. of
1x10 spores + crystals per bee over a 7 d period, no harmful effect was
noted.Honeybeesforaging treated areasarenotharmedby Btk use
(Bailey,1971 and Melin andCozzi,l990).
ii) Effect
on
silkworms: Btk strains consisting of CrylAa toxin are
recognizedto be toxic tosilkworm.A Btk strain in whichsporesare
inactivated, Toarow CT@ (Toagosei Co. Ltd.), is of low toxicity to the
silkworm (Navon, 1993).
iii) Effect on earthworms:Earthworms are of great importance in most
ecosystems especially in forests. It is therefore highly desirable that the
pesticides used should not endanger the earthworms. Various field and
laboratory studies indicate no toxic effects of Btk on earthworm. When
Btk at conc. of 30 g / M 2 is applied to small field plots, no adverse effects
on the earthworms population was seen and no dead or diseased worms
were found in the treatment areas evenafter 2 months.
In another field experimentwithanormalconcentrationof Btk
formulation(3600g/ha or about1 billion s ores/) and 100 times
higher concentration (about 1 trillion sporeshlP), no adverse effect of Btk
was found against earthworms within 7 weeks. During the experiment no
evidentdifference in the densityof snails, forficula, myriapodesand
woodlice was observed ( B e n and Altwegg, 1975 and Melin and Cozzi,
1990).As Btk is non-toxic to bees, andbecause it doesnotharm
predatory insects, it is ideally suited to integratedpestmanagement
programs.

3 Bacillus thuringiensis subsp. israelensis


Bti was isolated in 1976byGoldbergandMargalitfollowingscreeningof
various isolates from soil samples taken from known mosquito breeding sites in
Negev desert of Israel (Goldberg and Margalit, 1977). In 1978, H. de Barjac
determined the serotype designation for Bti as H-14 at Louis Pasteur Institute at
Paris using the standard flagellar technique. Bti was found to be fairly toxic to
dipteran
larvae (especially that ofNematocera),including
mosquitoes,
blackflies, homflies and stable flies inthe larval stage, but did not have any
adverse effect on animals, plants or other insects and was found to be non-toxic
to lepidopteran larvae. Mosquitoes and blackflies are not only bothersome but
also represent a serious risk to public health for they are vectors of a multitude
Bacterial 55

of diseases of man and animals through transmission of pathogenic viruses,


bacteria, protozoa and nematodes.
Thus, Bti has turned out to be of considerable importance especially in
the tropics, because of its potential as biological insecticide against Anopheles,
Aedes and Culex species and Simulium damnosum, the vectors of devastating
diseases such as malaria, yellow fever, filariasis and river blindness,
respectively.

3.1 Characteristics

Bti serotype H-14 is an aerobic, gram-positive, rod shaped,spore forming


bacteria. During sporulation, it synthesizes a cytoplasmic parasporal inclusion
body (or crystal). The Bti crystal is composed of at least four major protein
toxins, (a)Cry4A, 134 kDa (b) Cry4B, 128 kDa (c)Cry1 1A (formerly
designated asCryIVD), 72 kDa and (d) CytlA, 27 kDa and another minor
component CrylOA (formerly designated as CryIVC), 78kDa.The27 kDa
protein is responsible for cytolytic activity and the higher molecular mass
proteins are responsible for dipteran activity and nematocerous toxicity (Bozsik
et al., 1993).

3.2 Bti CrystalProteinsand Genes

3.2.1 Parasporal inclusion of Bti

Most of the mosquito-active strains of Bt produce spherical or irregular shaped


parasporal inclusions. The parasporal body of Bti is basically spherical and
averages about 1 pm in diameter, ranging from 0.7 to 1.2 pm(Figure 2.3).
The toxicity of Btis parasporal body varies considerably depending on
whether it is intact or solubilized and onhow it is assayed.Wheningested,
either intact or solubilized, the parasporal body is toxic to mosquitoes, black
flies, and several other nematocerous dipterans. In addition to being toxic to
mosquitoes, the solubilized parasporal body is cytolytic to a wide range of
vertebrate and invertebrate cells, including erythrocytes, and toxic to mice if
injected. Thus, Bti is characterized as being mosquitocidal, cytolytic, hemolytic,
and even neurotoxic. The cytolytic activity of the solubilized parasporal body is
attributed to a 25-kDa protein that is cleaved from the 27-kDa protein when the
parasporal body is solubilized under alkaline conditions(Thomasand Ellar,
1983 and Armstrong et al., 1985).
56 Chapter 2

Figure 2.3 Electron micrograph of the parasporal body of Bti developing sporc (Sp)
and parasporal body (PB) during sporulation;E, cxosporium (Source:
Fcderici et al., 1990).

Several subspecies of Bt other than israelensis have been reported to


produce parasporal bodies containing proteins toxic to mosquitoes. These
subspecies include morrisoni,
nledellin,
jegathesan,
kyushuensis,
darmstadiensis, shandongiensis, canadensis and galleriae. The parasporal body
produced by the isolate of Et subsp. morrisoni PG-14, contains same proteins as
Bti as well as an additional protein of 144 kDa (Federici et al., 1990 and Koni
and Ellar, 1994).

3.2.2 Bti encodinggenes

The Bti contains a 72-mDa plasmid from which several crystal protein genes
(c1y4A, cry4B, c ~ yOAl and cry1 I A ) and the cytlA gene have all been isolated.
The architecture of cry4A and cry4B is similar to that of the cryl genes. They
also encode ca. 130-kDa proteins, which are proteolytically converted into
smaller toxic fragments of 53-67 kDa(Chunjatupomchaiet al., 1988). The
crylOA gene encodes a protein with a predicted molecular mass of 78 kDa. The
cryl I A gene encodes a 72-kDa protein, which is a major component of the Bti
crystals (Federici et al., 1990). This crystal protein, unlike all other known cry-
encoded proteins, is proteolytically converted into an active fragment of ca. 30
kDa. The 27-kDa protein encoded by cytlA shows no sequence homology to the
other crystal protein genes (Hofte andWhiteley, 1989).
Bacterial Insecticides 57

3.3 Insecticidal
Activity

The Bti is insecticidal against at least 72 species of mosquitoes (Culicidae), at


least seven species of blackflies (Simulidae) and otherdipterans, including
chironomid larvae, sciarid flies and tipulids. Bti toxins have also been shown to
possess larvicidal activity against nematodes (Bone et
al., 1988). Upon
solubilization, the Bti toxin lyses erythrocytes and cultured mammalian cells as
well as cultured insect cells, whereas solubilized Lepidoptera-active toxins
display no corresponding cytotoxicity. Various species of mosquitoes have been
noted to exhibit different degrees of susceptibility toagivenpreparation or
formulation of Bti. In general, Culex mosquito larvae are more susceptible than
Aedes larvae, followed by Anopheles larvae (Mulla, 1986).
In Bti, three major anti-dipteran toxins ranging from 68 to 135 kDa
(Cry4A, Cry4B, and Cryl 1A) contribute to the overall toxicity of intact crystals
in a synergistic manner (Parket al., 1995). Cyt toxins are hemolytic and
cytolytic in vitro and are specifically active against dipteranlarvae in vivo
(Bozsiketal., 1993 and Guerchicoff et al.,1997).The CytlA toxin displays
greater capacity to synergize the action of the Cry4 and Cryl 1 toxins of Bti
(Hurley et al., 1987; Gill et al., 1992 and Poncet et al., 1995).
Some of the commercial products are based on two different strains of
Bti, i.e. VectoBac(Abbott) and Aquabac (BeckerMicrobial)basedonstrain
HD-507 and Teknar (Therm0Trilogy) based on strain HD-567.

3.3.1Factorsaffecting Bti activity

The efficacy of Bti in the field is influenced by many factors, such as species of
mosquitoes, vegetation cover, extent of water pollution, water flow rate, depth
of water, and prevalence of natural enemies.
The commercial formulations of Bti available now, can yield 95-100%
control of Culex, Aedes and Psorophora larvae at the field rates of 0.11 to 0.5
kg/ha of the primary products. Double and triple of these rates are necessary for
controlling Anopheles larvae. Rates of application have to be doubled or tripled
in treating deep, polluted or vegetated breeding sources (Mulla, 1986).
Various environmental factors such as temperature andpH of water
influence the efficacy of biolarvicide. Bti displays more activity at higher
temperature against Anopheles spp. and Culex spp. The activity enhancement for
Anopheles stephensi and Anopheles culicifacies from 2 1C to 31C was found to
be of the order of 3- and 5-fold respectively (Mittal et al., 1993a) (Table 2.3).
On the other hand, at high water pH effectiveness of Bti is greatly reduced
(Table 2.4).
58 Chapter 2

Table 2.3 Effect of tempcrature on the actwityof Bti against Atzopheles spp.
(Adapted from Mittal et al., 1993).

LC50 Values (nlg/l) after 40 hr exposure


Tenlperature Anopheles stephetlsi Anopheles
culicifacies
"C 31 ?I 31 ?I
LC50 LC50
LC50 /LC50 LC50/LcSO

21* 0.25 0.8


31" 0.076 0.17
3.3 4.7

3.4 Mode of Action

The mode of action of Bt is given in detail in Section 6.0 of Chapter 1. The Cyt
toxin is a unique component of Bti that does not share homology with the Cry
toxins. The binding receptors are different for Cry and Cyt toxins. While Cry
toxin binds to midgut glycoprotein receptor, the Cyt toxin bind to unsaturated
phospholipid.Althoughbothtoxinsshare a commoncytolyticmechanism
involving colloid-osmotic lysis, the actual mechanism of pore formation may be
different. The gut of mosquito larvae is a simple cuboidal epithelium, with no
goblet cells. The Bti toxin mainly affects the posterior zone of the midgut of
dipteran larvae, as against Btk toxin that affects anterior zone of the midgut of
lepidopteran larvae (Lakhim-Tsror et al., 1983; Knowles and Ellar, 1987; Gill et
al., 1992; and Knowles, 1994).

Table 2.4 Effcct of waterpH on the larvicidalactivity of Bti (Source: Malaria


Research Center, New Delhi, 1993,Progress Report of biolarvlcidcs
in vector control)

Percent mortality of nlosqrritoes


Mosquito Concentratiot1 PH
species n1gA
3.5 7.5 9.5 10.5

Anopheles steplletlsi 0.5 78 90 94 8

Culex quirlquefasciatus 0.25 100 98 88 12


Bacterial Insecticides 59

3.5 Persistence

Bti is compatible with the environment and is particularly suited for application
in areas that are experiencing resistance development among the target species
to organophosphate larvicides. It does not persist insoil or in aquatic
environments and its residual activity and persistence in the environment are
limited to only a few days. Over time, the Bti crystals gradually settle to the
bottom of aquatic environments where they become inactivated through binding
with soil particles or are used as a protein source by other non-target organisms.

3.6 Safety and EcotoxicologicalEffects

After more than a decade of use, Bti remains a safe alternative to chemical
pesticides used in the aquatic environment. No adverse incidents have ever been
reported following its use and there have never beenany toxic incidents
involving fish, wildlife or man.

3.6.1 Mammalian safety

The toxicological studies of Bti were performed by experimental exposure of


various animals (mice, rats, guinea pigs, and rabbits) and indicated the absence
of acute and prolonged toxicity. All acute toxicity tests gave negative results
through a variety of pathways - subcutaneous and intra-peritoneal injection,
gavage, inhalation, percutaneous applications, scarification of the skin, and
ocular inoculations - using mean doses of about lo7 to lo8 bacteria per animal.
There was also no anaphylactic shock obtained in guinea pigs, and successive
passages in mice of Bti did not induce any virulence. In investigating sub-acute
oral toxicity in mice and rats, through repeated adrmnistration of Bti for a period
of three weeks, during which the equivalent of about 10 to 10 bacteria per
animal was ingested, results were negative. These studies in mice, rats, guinea
pigs, and rabbits confirmed that mammals are highly tolerant of the organism.
Animals exposed to Bti rapidly eliminated the organism from their systems and
tissues showed no evidence of multiplication. In summary, neither pathological
symptoms, nor diseases, nor mortality have been
observed (Siege1 and
Shadduck, 1990a). Bti has also been shown to be safe to amphibians and data
indicate that these organisms would not be affected atoperationaldosages
(Paulov, 1985).
All the published data indicate that Bti is not asignificant toxicant
using conventional routes of exposure. Thus, Bti is avirulent and non-pathogenic
and can be safely used in environments in which human exposure is likely to
Chapter 2

occur including exposure to immuno-deficient humans (Siegel, Shadduck and


Szabo, 1987 and Siegel and Shadduck, 1990b).

3.6.2 Effect on non-targetorganisms

Bti is considered environmentally benign because testsonmost non-target


aquatic organisms have not resulted in discovery of deleterious effects.
However, several families of Nematocera besides CulicidaeandSimuliidae,
such as the non-target Blephariceridae and some Chironomidae, are susceptible
to Bti (Painter et al., 1996).
Selectivity in target and non-target nematoceran Diptera is the result of
a combination of filter-feeding behavior, alkaline gut pH, andpresence of
certain proteolytic enzymes in the gut that activate the toxin. These factors
enable the capture, dissolution, and activation of the toxin containing parasporal
inclusion bodies (Lacey and Mulla, 1990). The absence of any of these factors
greatly reduces or nullifies the susceptibility of a given speciesto the toxic
effects of Bti. For example, filter feeding Ephemeroptera, Trichoptera,and
others may actually ingest the toxin, but without subsequent activation by pH
and proteolytic enzymes, the crystal remains harmless to these organisms. Some
Nematocera maybe susceptible but their method of feeding(eg.predator,
scraper, gatherer, etc.) reduces the amount ingested and hence the exposure is
probably below toxic limits (Cibulsky and Fusco, 1987).
It has been found that Bti has no noticeable adverse effects on Odonate
naiads, dragonflyand damselfly naiads (Ali and Mulla, 1987). Bti minimally
affects predacious mosquito species of the genus toxorhynchites. There were no
adverse effects on crustacea by the operational dosages of Bti.
Thus, from the data obtained in laboratory and field tests, it can be said
that the use of Bti has minimal if any impact toward non-target organisms in the
aquatic environment.

4 Bacillus sphaericus
Bacillussphaericus, a cogeneric species of Bti, alsoproducesparasporal
proteinaceous toxin crystals during sporulation, which aregenerally active
against Culex and Anopheles species. Kellen and Meyers first isolated in 1964, a
Bacillus sphaericus strain active against mosquito larvae from moribund fourth
instar larvae of Culiseta incidens. Kellen's strain was later designated as strain
K. This strain had a low order of larvicidal activity because of which it could not
be used in mosquito control. The identification of the strain SSII-1 in India by
Samuel Singer in 1973, renewed interest in B. sphaericus. But only after the
isolation of strain 1593 in Indonesia from dead mosquito larvae, which exhibited
Bacterial 61

a much higher level of mosquitocidal activity, was the potential ofB. sphaericus
asabiologicalcontrolagenttakenseriously.In 1980, Wickremesingheand
Mendis isolated what they called strainMR-4. Thls strain has since been given
the designation2297. This strain along with strains 1593 and 2362, remain some
of the principal candidates of field interest among a growing 30-50 list ofstrains
of B. sphaericus thatareavailableforuse(Singer, 1990 and Charles et al.,
1996). B. sphaericus has been found to be innocuous to mammals. It also shows
a recycling potential under field conditions. In addition, with the discovery of
increasingly toxic strains, this biological insecticide seems very promising for
control of mosquitoes.

4.I Characteristics

Bacillus sphaericus is a gram-positive, mesophilic, strictly aerobic, round spore


former saprophybc bacterium (Figure2.4). The spore size is around0.5-1.0 pm.
B.sphaericus is foundcommonlythroughouttheworldinsoilandaquatic
environments.
B. sphaericus toxinremainsextremelystableunderoptimalstorage
conditions of neutralpH and4C and fairly stableat room temperature, however

Figure 2.4a Scanning micrograph of B. sphaerinrs 1593 (Kim and Lee, 1984).
62 Chapter 2

Figure 2.4b Electron micrograph showing crystal protein in a sporulated cell


of Bacillus sphaericus (Charles et al., 1988)

a steady decline in larvicidal activity of a primary powder occurs when stored


out of adesiccatorandsubjectedtotemperatures 13-30' C. Toxinthatis
exposed to high pH(10.8) will become denatured immediately. A rapid decline
in spore viability, on the other hand, is observed during storage under acidic
conditions. Although viable spores are not required for larvicidal activity, they
are required for persistence of activity due to recycling (Lacey, 1990).
These bacteria are also characterized by negative reaction to most of
the traditional phenotypical testsused for the classification and identificationof
bacilli. This largely arises from their obligately aerobic physiology and inability
to use sugars as source of carbon and energy. The findings of various genetic
and
biochemicalstudies
indicate
that
phenotypically B. sphaericus is
heterogeneous. Both insecticidal (1593,2362,2297) and non-insecticidal (SSII-
I) strains ofB. sphaericus are known (Russell etal., 1989 and deBajac, 1990).

4.2 Toxins Produced by B. sphaericus

The entomocidal B. sphaericus strains synthesize two types of toxins, binary


toxin(Btx)andmosquitocidaltoxin(Mtx).Manyhigh-toxicitystrains
synthesize both Btx andtheMtx,whileotherssynthesizeonlythebinary
toxin. Low-toxicity strains synthesize either none of the two toxins or only the
Mtx. Boththetoxinsdifferintheircompositionsandtimeofsynthesis.
Thereis also evidence for presence of additional toxin genes encoding novel
type of mosquitocidal toxins of 31.8 kDa and 35.8 kDa (Liu et al., 1996 and
Thanabalu and Porter, 1996).
Bacterial 63

4.2.1 Binaty
toxin
(Btx)

The binary toxin is present in all highly active strains and accumulates during
the early stages of sporulation and forms a small crystal in the mother cell. The
crystal comprises of equimolar amounts of two proteins of 4 1.9 and 5 1.4 kDa,
often referred to as the 42 and 51-kDa toxin respectively (Baumann etal., 1991).
Both the proteins are needed for larval toxicity. Strains which synthesize the
binary toxin are referred to as high toxicity strain because of the acute toxicity
conferred by thelarge amounts of proteinin the crystal (Priest etal., 1997).
Theamino-acidsequencesof42 kDa and 51 kDaproteinare not
similar to those of any other bacterial toxins, including those produced by Bti.
Therefore, they constitute a separate family of insecticidal toxins. Subcloning
experiments have shown that 42 kDa protein alone is toxic for mosquito larvae
Culex pipiens, although the activity is weaker than that of crystal containing
both proteins. In contrast 51 kDa protein alone is not toxic, but its presence
enhances the larvicidal activity of 42 kDa protein, suggesting synergy between
the polypeptides.Thelargerprotein is postulated to actasabindingprotein
enabling the entry of the 42 kDa protein into the midgut cells of the larval gut.
(Nicholas et al., 1993 and Charles et al., 1996).

4.2.2 Mosquitocidal
toxin (Mtx)

Mosquitocidaltoxin(Mtx) is unrelated to Btx. It is synthesizedduring


vegetative growth phase and is proteolytically degraded as the cells enter the
stationary phase. Three types of Mtx toxins have been described, Mtxl, Mtx2
and Mtx3with molecular mass of 100,30.8 and 35 kDarespectively.These
toxins do not display any similarity to each other
or to the crystal proteins or any
other insecticidal proteins (Baumann et al., 1991).

4.3 Classification

With the increasingnumberofisolatedstrains of B.sphaericus, it became


apparent that there isa need to differentiate among the strains,asdifficulty
arises in their identification and differentiation- first among the atoxic andtoxic
strain and secondfromeachother within the toxicgroup.Thereare few
phenotypic differences between atoxic and toxic strain other than insecticidal
activity.Moreover,thephenotypiccharacterizationthatisusedin Bacillus
taxonomy and which has proven useful for differentiation of B. thuringiensis
strains, fail to distinguish betweenB. sphaericus isolates.
Numerous methods have been tried to classify this very heterogenic
species.Classification via bacteriophagetyping(Yousten,1984) and flagellar
64 Chapter 2

(H) serotyping (de Barjac, 1990),similar to that for B. thuringiensis strains, have
been used. In early studies, toxicity and serotypes or phage-types appeared to
correlate, high toxicity strains were classified largely in serotype 5a5b, phage-
group 3; serotype6,phage-group3andserotype 25, phagegroup4.Low
toxicity strains were classified in serotypes la, phage-group 1. Strains showing
an average toxicity were classified in serotype H26a26b, and serotype H2a2b,
phage-group2.Theexception to correlation is the phage-group4with less-
toxic strains 2173and2377andhighly toxic 2297.Thediscovery of high-
toxicity strains from Ghana belonging to serotypes 3, 6 and 48 conksed the
supposed correlation between serotypes and suggested that the relationships may
not be so straightforward (Table 2.5) (de Barjac, 1990 and Priest et al., 1997).
Some of the recently developed techniques including numerical classification,
ribotyping, cellular fatty acidcompositionanalysisandrandom amplified
polymorphic DNA analysishavebeenused for the classification which also
indicate that mostof the pathogenicgroups are recovered in afewgroups
(Charles et al., 1996).
Binary toxin genes were detected in chromosonal DNA of strains from
serotypes la, 3, 5a5b, 6, 25 and 48 but not in strains from serotypes 2a2b, 9a9c
or 26a26b (Table 2.5). Mosquitocidal toxin genes were detected in strains from
serotypes la, 2a2b, 5a5b, 6,9a9b, 25 and 48 and is absent fiom strains of several
serotypes, notably all those of serotype 3 and most of serotype 2a2b. Overall
there was little correlationbetween the serotypeandtoxingene distribution
except for serotype 5a5b strains. Strains with serotypes la, 2a2b, 3, 6,25 and 48,
all showedheterogeneityof the toxicity genotype to variousdegrees.Only
strains ofserotypes5a5b(highly toxic) and26a26b(weakly toxic) were
consistent in their toxicity pattern. Thus, although serotyping provides a useful
framework for strain identification, its reliability as a predictive tool must be
treated cautiously (Priest et al., 1997).

4.4 Insecticidal
Activity

B. sphaericus formulations have been reported to be highly insecticidal against


larvae of Culex and certain species of Anopheles but not against Aedes aegypti.
The toxicity of B. sphaericus, particularly against larvae of Anopheles species,
isgenerally low as compared to that of Bti, and the mortality is obtained in 48 to
72 hrs. But it has been reported to persist and recycle in the treated habitats.
However, B. sphaericus has greater biological activity than Bti against Culex
larvae, producing 95100% control of Culex larvae at the rates of 0.1 to 0.2
kg/ha in clear water. This rate of application has to be increased in polluted,
vegetated or deeper bodies of water (Davidson al.,et 1981 and Mulla, 1986).
Bacterial 65

Table 2.5 Classification pattern and distribution of Btxand Mtx genes in B.


sphaericus (Ref.: de Barjac, 1990; Charles etal., 1996; Zahner and
Priest, 1997 and Prlest et al., 1997)

Straill Origin Btx g e m Mtx gene Phage type sero type

K United States No Yes 1 la


SSII-I India No Yes 2 2a2b
1889 Israel No Yes 2 2a2b
1883 Israel No Yes 2a2b
IAB 88 1 Ghana YCS No NR 3
LPI-G Singapore Yes No 8 3
1593 India Yes Yes 3 5a5b
1691 El Salvador Yes Yes 3 5a5b
2013.6 Romania Yes Yes 3 5a5b
2362 Nigeria Yes Yes 3 5a5b
2500 Thailand Yes Yes 3 5a5b
BSE 18 Scotland Yes Yes 3 5a5b
1593M Indonesia 3 Sa5b
IAB 59 Ghana Yes Yes 3 6
COK 3 1 Turkey No YCS 8 9a9c
2297 Sri Lanka Yes Yes 4 25
2173 India No No 4 26a26b
2377 Indonesia No No 4 26a26b
IAB 872 Ghana Yes Yes 3 48

The larvicidal activity of the microbial agent depends not only on the
strains intrinsic properties, but also on the species of mosquito and can thus
vary within a genus. Toxicity levels vary among the larvicidal serotypes and
even within the same serotype (de Barjac,1990).
A few commercial products based on B. sphaericus strains are listed in
Table 2.6.
66 Chapter 2

Table 2. 6 Somecommercialformulationsof B.sphaerlcus

Trade Stram Manrcjacturer Name

B-101 Biological
Preparations,
Spherix
Berdsk, Russla
imos 2362 Biochem Products
362 VectoLex Abbott Labs

4.4.I High temperatures enhance toxicit),

The toxicity of Bacillus sphaericus against Anopheles larvae was found to get
enhancedconsiderablyat higher temperatureTable 2.7). A B.sphaericus
formulation in a laboratory study showed a very large enhancement in toxicityat
31C ascompared to toxicity at 21OC; anearly250-foldincreaseagainst
Anopheles stephensi and 100 fold increase against Anopheles culicifacies (Mittal
et al., 1993a). Earlier, Subramoniametal.(1981) had reportedasimilar
observation of activity enhancement of B. sphaericus against An. subpictus. In
comparison,a Bti formulationshoweda relatively modestenhancementin
toxicitywithtemperature(seeTable2.3).Theeffect of temperature on the
activity of B. sphaericus against Culex species was relatively modest.
It is known that B.sphaericus toxin binds to midgut cells of Anopheles
larvae with low affinity as compared to Culex larvae, where it binds with high
affinity. Perhaps, at higher temperature, in additionto enhancement of larval

Table 2.7 Effect of temperature on the activity of Bacillus sphaerrcus


(Adapted from Mittal et al., 1993a)

Tenlperaiure LCJovalues (tng/l) after 40 hr exposure

Anopheles
stephensi
Anopheles
culicifacies
"C
21 31 31 21

0/LC50LC50 /LC50 LC50 LC50

21" 10.2
0.48 3 1' 0.04
255 >IO0
Bacterial 67

feeding rate, binding characteristics of 5.sphaericus to larval midgut cells of


Anopheles species also improve significantly (Figure 2.5).

4.5 Mode of Action

Following ingestion of the spore-crystal complex by a susceptible larva, the


protein-crystal matrix quickly dissolves in the lumen of anteriorstomach
through the combined action of midgut proteolytic enzymes and high pH. It is
followed by activation of the 5 1 and 42 kDa toxin of the binary toxin to 43 and
39 kDa toxins respectively, and binding of the activatedtoxinsto specific
receptor molecules. The role of the N-terminal region of the 51 kDa is to direct
the binding of the toxins to specific receptor molecules, and its C terminus is
necessary to interact with the 42 kDa toxin. 5. sphaericus releases the toxin in
all species, evenin non-susceptible species such as Ae. aegypti. Midgut
alteration starts as soon as 15 min. after ingestion. 5. sphaericus toxins cause
disruption in midgut epithelial cells, specifically in the regions of gastric caecae
and posterior midgut. This leads to mortality within 4 - 48 hr after treatment,
depending on doses ingested. However, the mode of action of Mtx toxins is still
unknown (Charles et al., 1986).
Studies have identified the various factors that are responsible for
differences in susceptibility among mosquitoes to B. sphaericus. The lack of
susceptibility in Aedes mosquitoes (especially Ae. aegypti) to B. sphaericus is
not due to rates of toxin ingestion, dissolution of the toxin, or specificity of the
gut proteases, but is due to failure of toxins to bind to midgut epithelial cells,

Toxin bindingto
midgut cells of
Moderate larvae

Culex
B. sphaericus
Effect of

on activity

High

Figure 2.5 Effectof temperature on toxin binding and activity of


B. sphaericus
68 Chapter 2

since no functional receptors were found in this species. For Anopheles species,
B. sphaericus toxins bind to larval midgut cells butwith lowspecificityand
affinity, thus resulting in moderate degree of susceptibility. In Culex species the
toxinsbind with highspecificity and highaffinity,thusleading to high
susceptibility in these species (Rodcharoen and Mulla, 1995).

4.6 Persistence

Field trial experiments have demonstrated that B. sphaericus has recycling and
persistence ability. This may be due to recycling and multiplication of spores in
larval cadavers and certain aquatic situations ormay be simply due to long-term
persistence of sufficient and accessibletoxin or acombination of both
(Davidson et al., 1984; Des Rochersand Garcla, 1984). However, the activity
andpersistence of B. sphaericw depends upon the type of strain, its
fermentation and formulation methods and also on environmental factors such as
water quality and depth, solar radiation,target species, and larval density.
B. sphnericus can apparently persist in a variety of habitats, including
those that are polluted. In shallow containers, in clear water, larvicidal activity
has been reported as persisting for nine months. In some extremely enriched
andor deep breeding sites, however, curtailment of larvicidal activity has been
reported. Ostensibly, toxin either settled out of the feeding zone of the larvae or
ingestion of sufficientquantities wasinhibited by excessparticulatematter
(Lacey, 1990).
InlaboratorystudieswiththeSSII-1isolate,Davidsonetal.(1975)
reported the germination, growth and sporulation of B. sphaericus in cadavers of
Cx.quinquefasciatus with a subsequent log increase in spores over that ingested
by the larvae. Hertlien et al. (1979) retrieved the viable and infective spores of
the 1593isolateseveral months afterintroductionintolarvalhabitats.They
concluded that B. sphaericus is capable of recyclingin nature.
Various studies indicate that, under certain conditions, larva to larva
recyclingoccursand may perpetuateeffectivecontrolforseveralmonths.
Amplificationsof B. sphaericus in larvae may providesignificantrecycling
under field conditions when large numbers of susceptible larvae are present at
thetime of treatment. Larval densityappears to beanimportantfactor in
determining the occurrence of recycling of B. sphaericus and hence its effective
duration of activity. Larval numbers, in turn may be afinction of species andor
environmental conditions (Lacey, 1990).
Although the recyclingof B. sphaericus may not provide enough spores
for long term vector control, the implication that subsequent application dosages
in same locality can be lower should be taken into consideration when cost-
effectivenessofusing B. sphaericus for vector control is deliberated(Yap,
1990).
Bacterial 69

4.7 SafetyandEcotoxicologicalEffects

B. sphaericus is quite specific, impacting mosquito larvae only, with no adverse


effects on mosquito predators. The LCso value of Spherix (a formulated product
of B.sphaericus strain B-101, serotype H5a5b) against larvivorousfish
Gambusia affinis and Poecilia reticulata and also frog tadpoles and
Mesocyclops is reported to be greater than 1 g/l. Thisconcentration is
approximately 500 and 20,000 times more than that required for effective
activity against larvae of An. stephensi and Cx. quinquefasciatus respectively.
Similarly, the LCso values against notonectid bugs, Enithares indica and Anisops
sardea is found to be greater than 100 mg/l and 50 mg/l respectively, which are
also approximately 500 and 250 times higher than in An. stephensi larvae (Mittal
et al., 1993b). B. sphaericus is non-toxic to most non-target organisms (Mulla,
1986) and has shown no mammalian toxicity, is safe to fish and wildlife and
practically all macro-invertebrate associates of mosquito larvae.

4.8 Comparisotr of Cltaracteristics of Bti and B. sphaericus

Bti and Bacillus sphaericus are two gram-positive soil bacteria that have been
successfully used for biological control of blackflies and Culex mosquitoes.
However, both Bti and B. sphaericus crystals differ in toxin composition, modes
of action, and insecticidal spectra (Table 2.8) (Priest, 1992; Porter et al., 1993
and Porter, 1996).

5 Concluding Remarks
Btk is widely used as a microbial insecticide against lepidopteran pests. Due to
its specificity, Btk does not disrupt the natural balancebetweenpestsand
beneficials. This is of particular advantage for forest and other control programs
where it is desirable to maintain a natural balance of beneficials to suppress the
resurgence of damaging insects. The short half life of B. thuringiensis in the
field is generally believed to minimize its impact on non-target organisms. On
agricultural crops, most of the activity of Btk towards target Lepidoptera
disappears within 3-4 days after application. In addition to the development of
Btk isolates as conventional insecticides, transconjugate and
genetically
engineered strain with novel properties are increasingly coming to the market
(see Chapter 3).
Efforts to reduce the populations of dipteran species which transmit
tropical diseases such as malaria, filariasis, and onchocerciasis through naturally
occurring entomopathogenic bacterial strains, has beenreceiving increased
attention. Over the last two decades, there has been much interest in the toxins
70 Chapter 2

Table 2.8 Comparisonofcharacteristicsof Bfi and B. sphaericus

sphaericus
Characteristics B. Bt-lsraelensis

Toxin B f i incluslons are made of B. sphaericus inclusions are made


Composition four major protelns, Cry4A, of equimolar amount of proteins of
Cry4B, Cry1 1 A and Cyt 1A. 42- and 5 1 kDa which act as a
binary toxin.

Protoxin crystals produced Protoxin surroundedby the


durmg sporulation arc cxsporium membrane.
deposited along sidc the
spore.

Mode of actlon B f i toxin completely breaks B. sphaericus toxin does not break
down the larval midgut down completely.
epithelium.

lnsccticldal Bfi is toxic to Aedes, Cuku, Target spectrum is restricted. It is


spcctra Attoplreles, Matrsottia aud toxic to Culex and Anopheles and
Sitwliunl larvae. is poorly or non-toxicto Aedes
larvae.
Pathogenlc to blackfly larvae. Not pathogenic to blackfly larvae.

Effect of Activity cnhancement against Very large activity enhancement


temperature Anopheles and Culex larvae against Anopheline spp. at higher
on activity at higher temperatures. temperatures.

Persistence and Bti does not perslst for long More persistent than that ofBti,
Recycling periods in the environment, including in polluted water.
especially in polluted water.

Bfi does not recycle. B. sphaericus can recycle under


certain environmcntal conditions.

Bfi crystals are not linked to Toxic agents are closely linkedto
the spores and they settle spore which would then function
more rapidly than B. as a kmd offloat.
sphaericus.

Resistance Resistance has not been Potential for development of


observed even after years of resistance to binary toxin.
intensive field treatments,
probably because of multi-
component structure of the
crystal.
Bacterial 71

of Bti and B. sphaericus for mosquito larval control. Their high specificity for
their target mosquitoeshas an important practical consequence. Bti canbe
regarded, as a natural alternative to chemical insecticides for mosquito control as
it is harmless for the non-target organismsof the water fauna. In practice,Bti has
been used as a replacement for chemical insecticides whenever they have been
banned or where insecticide resistance has developed. Bti has been widely used
in West Afrlca in a successful vector
compaign(the
World Health
Organization'sOnchocerciasisControl Program) against blackflies, Simulium
damnosurn Theobald. There are some reports of Bti use in integrated control
programs e.g. of mosquitoes, in conpnction with larvivorous fish (see Chapter
10) andother natural predators. The lack of anyseriousresistance to Bti in
mosquitoes and blackflies has been interpreted as possibly due to complexity of
the multi-toxin (Cry4 and Cry1 1) inclusions (see Chapter 5). The development
of B.sphaericus as a microbial control agent of mosquitoes has been somewhat
hamperedby the successandbroaderspectrum of Bti. Despite that, B.
sphaericus offerssome distinct advantagesover its commerciallyproduced
counterpart. Most notable is the increased duration of larvicidal activity against
certainspeciesofmosquitoesespecially in organicallyenriched(polluted)
habitats. However, the future impact of Bacillus sphaericus will depend largely
on its ability to be more persistent, its recycling ability in field trials, and its
possibility for use as a microbial control agent in malaria control programs.

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76 Chapter 2

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Genetically Modified Bt Strains
and Bt Transgenic Plants
1 Introduction
The importance ofBt in an environmentally soundinsect control program is well
accepted. Therefore there is an active interest in search for novel toxins as well
as in the discovery of moreactive strains. The bacterial strains used for all ofthe
early and some of the current Bt foliar insecticides are wild type strains, that is,
they were found in nature in the form in which they are used to produce the
microbial spray. The use of conventionalBt insecticides, however has limitation
like narrow specificity, lowpotencyand short shelf life. On the otherhand,
certain combinations of Cry proteins have been shown to exhibit synergistic
effects. Therefore, genetic manipulation ofBt - to create combinations of genes
more usefid for a given application than thoseknown to occur in natural isolates
- maybe desirable. Thesecombinationscanbeconstructedbyvarious
approaches that utilize the tools of molecular biology and genetic engineering,
as well as conventional microbiological methods.
Severalreviewshaveappeared inthe literature coveringgenetically
modified Bt strains (Baum et al., 1999;Cannon,1996;Kumar et al., 1996;
Carlton and Gawron-Burke, 1993; and Koziel et al., 1993b) and transgenic Bt
plants (Huang et al., 1999; Jenkins, 1999; Schuleret al., 1998; Peferoen, 1997

77
78 Chapter 3

and Estruch et al., 1997). This chapter provides an overview of the


developments in both genetically modified Bt strains and Bt crop plants.

2 Novel Bt Strains ThroughConventionalGenetic


Techniques
The manipulation of Bt at the molecular level requires techniques for the
efficient movement of DNA into and among Bt strains. Several methods have
been used to move DNA between Bt strains and from heterologous species. It is
possible to construct strains with increased activity on specific pests by using
classical genetic techniques to eliminate unnecessary &endotoxinsand / or
introduce desirable ones.
Multiple &endotoxin genes could be introduced into a strain for one or
more of the following reasons:
(a) To increase the toxicity on a particular pest,
(b) To broaden the host range in cases where one&endotoxincan not be
identified with acceptable toxicity on multiple insect targets, and
(c)To manage resistance by including &endotoxins that bind to different
sites in the insect midgut.
However, the effectiveness of the strain will depend on the biosynthetic
capacity of the cell, because introducing more toxins to broaden specificity will
usually reduce the amount of each&endotoxinpresent. Regardless of the
technique used for rational strain construction, the first and perhaps the most
difficult step is to determine the desired insecticidal activity and to identify 6-
endotoxins with these properties.
Bt strains present in several currently available bioinsecticides were
constructed using the following techniques.

2.1 Conjugdolt

Bacillus thuringiensis in nature generally contain several different genes that


endow different insecticidal activities. Bt cry genes usually reside on specific
non-essential DNA molecules termed plasmids, although some are
chromosomally located also. Plasmids can either be removed (cured) from cells
that harbor them or they can be transferred between different strains by a natural
mating process called conjugal transfer. It occurs in nature between Bt strains
and can be replicated in the laboratory producing new Bt strains with different
spectra of toxicity.
Ecogen Inc., a U S . Biotechnology company has been actively involved
in development of trans-conjugate strains of Bt with novel properties that have
Genetically Modified Bf Strains 79

higher levels ofeffectivenessand that canactagainst a broaderarrayof


significant insect pests than wild-Bt strains. For instance, they combined beetle-
active with caterpillar-active Bt proteins in the same strain by conjugating Bt-
kurstaki with Bt-tenebrionistoproduce a hybridstain EG 2424(Foil@). It
contains a unique combination of coleopteran- and lepidopteran-active Bt ICPs
(CrylAc (2) and Cry3A) and resultantly has an expanded host range to control
Coloradopotatobeetle in potatoesandEuropeancornborer(Carltonand
Gawron-Burke, 1993), However, Ecogen has discontinued Foil@xroduct.
Similarlyothercommercialproductssuch as CutlassandCondor@,
whichcombine the insecticidal properties of bothparentstrains,havebeen
produced by conjugationprocess.For their construction, a self-transmissible
CIYIA plasmid was transferred via conjugation from a Bt-aizawai strain to a Bt-
kurstaki recipient strain. On the otherhand, in the products such as Agree@ and
Design', a cry1 plasmidfrom a Bt-kurstaki strainwastransferred to a Bt-
aizawai recipient strain (Baum et al., 1999). The active ingredient of Agree@
consists CrylAa,
of CrylAc, CrylCand
CrylD proteins.
Various
commercializedtransconjugate Bt productsand the range of insect pests
controlled by them aregiven in Table 3.1.
However, the conjugationalapproachtocreate novel Btstrainshas
certain limitations. Notall Bt toxin genes are located on transferable plasmids.

Table 3.1 Commercialtransconjugate Bt products

Bt recipient Conjugative Trade


Strairl Plasmid Name
Conzpany Target

Btk Bt-aizawui Condor Ecogen Soybean looper, velvct bean


(EG 2348) caterpillar, green clover-worm,
gypsymoth and spruce budworm.
Btk Bt-aizawui Cutlass Ecogen Beet armyworm cabbage looper,
(EG 2371) diamondback moth and cabbage
web worm.

Bt-aizawui Btk Agree Thermo Lepidoptera, especially


(GC-91) Trilogy diamondback moth.

Bt-~i~awui Bt Design Thermo Lepidoptera, especially


Trilogy diamondback moth.

Btk Bt-tertebr~orlis Foil* Ecogen Colorado potato beetle, European


(HD-263) (EG 2424) corn borcr armyworm and looper.

* Discontinued
80 Chapter 3

Second, the toxin protein with useful insecticidal activity may be synthesized at
low amount. Plasmid incompatibility could also be a problem. Conjugation is
not easily controllable in the laboratory and therefore it limits the number of
toxins (generally only 2-3)that can be presentin the final strain.

2.2 Electroporation

Electroporation technique offers another approach of genetic transformation of


Bt cells. Througha short electrical discharge,atemporaryand reversible
breakdown of the cell membrane is achieved,whichallowshighmolecular
weight substances such as DNA to enter the cell..This enables the introduction
of cloned toxic genes back into various Bt strains. For example, aBt-tenebrionis
gene was introduced into Bt-israelensis, resulting in a dipteran-active strain with
additional activity against Pieris brassicae, apropertywhichneitherparent
strain possessed (Crickmore etal., 1990).

2.3 Transduction

Transduction is the transfer of bacterial DNAbetween cells (intra- or inter-


serotype) via transducingphage particles, atechnique that is useful for both
gene mapping and the production of recombinant strains. Kalman et al. (1995)
usedatwostepprocedure to placea crylCa genefrom Bt-aizawai into the
chromosomes of two Bt-kurstaki strains, which contained multiple cry genes. In
the first step, an integration vector was usedtoplace crylCa gene into the
chromosome of Bt-kurstaki HD-73 via electroporation. In the secondstep,a
generalizedtransducingbacteriophagewasused to transfer the integrated
clylCa gene from Btk HD-73 to two other Btk strains, thus producing strains
with a broaderinsecticidal spectrum.

2.4 Classical Mutation

High-energy radiation hasprovedtobeoneof the most effective modes of


mutagenizinggenes.NovoNordiskused classical mutationtoimprove Bt-
tenebrionis. This strain produces a bigger crystal, which is directly correlated
withenhanced field activity. ThecommercialproductNovodor@(Abbott) is
based on amutant Bt-tenebrionis strain NB 176 containing two or three copies of
Cry3A and is obtained by gamma irradiation of Bt-tenebrionis strain NB125. It
produces unusually large rhomboid crystals composed primarily of Cry3A toxin
(Gurtler and Petersen, 1991).
ModifiedGenetically Bt Strains 81

Although these genetic manipulation techniques have the capability to


increase potency and control specificity, they are limited by several factors due
to which it is not possible to construct a strain containing only the desired 6-
endotoxins genes. The tools of genetic engineering can be used to overcome all
of these limitations.

3 Recombinant DNA Technology


The possibility of transferring specific genes from onecell to another could give
rise to a wide array of new products with desired characteristics. The discovery
of restriction enzymesas molecular scissorsto cut DNA into reproducible
fragments at specific sites has been used to create new moleculesby splicing or
recombining two different pieces of DNA. Restriction enzymes can be used to
cut specific sequences of DNA (genes) and to cut open a bacteriums plasmid
(cloning vector). The cut ends of the gene and the cut ends of the plasmid are
chemically sticky, so they attach to each other (recombine), to form a new
circular plasmid containingthe new gene (Figure3.1). The recombinant plasmid
carriesgeneticinstructionsfor the production of anewproteinandwhen
inserted into a bacterium,it produces this new protein.

Bacterium
Chromosom
Plasmid is A cut plasmid
NA removed
from

DNA animal cell

0 4 L1
DNA is Restridion
The cut ends
theof
DNA in cell removedenzymes plasmids and thecut
nudeus from Cell cutopen the ends of the new genes
nudeus plasmidand are chemically sticky
cut out a so they All attach to
gene from -
each other recombine
the DNA of - to form a new loop
containing another the
gene inserted organism

Figure 3.1 Recombinant DNA technology.


82 Chapter 3

Recombinant DNA technology provides the tools for developing safe,


efficientandcost-effectivemicrobialcontrolagents. Itis now possible to
combinethebesttraits of severaldifferentorganismsintoasinglestrain,
expressing&endotoxins that exhlbitenhancedinsecticidalactivity,longer
residual activity and broader host range. Modified Cry proteins engineered for
improvedproductionortoxicitycanalsonowbereadilyused as active
ingredients.
Conjugation and transduction have been used to transfer
recombinantplasmidsfromdonor to arecipient Bt; howeverthepreferred
method of gene transfer employs the use of electoporation, for which numerous
protocols are available (Baum et al.,1999).
A number of convenientshuttlevectorsfunctionalin E. coli and
Bacillus species have been constructed using replication origins from resident Bt
plasmid. These shuttle vectors, which exhibit good segregational stability, are
employed to introduce new toxinBt genes to Bt strains (Figure3.2).

ICP gene Bt Cloning


vector
Spore

........
Recombinant

;c-=
ICP Plasmid

DNA DNA

..........
........... ,,,Oo@
................... ...........

Recombinant Bacterium

Figure 3.2 Construction of genetically improved Bt strain.


Modified
Genetically Bt Strains 83

Alternatively, integrational vectorshavebeen used to insert cry genesby


homologous recombination (recombination between homologous segments on
different DNAs) into resident plasmids orthe chromosome.
A new Bt- based plasmid vector system enabled the introduction of a
cry3A gene into Btk (HD-119) without affecting the expression levels of the
native cry genes (Game1 and Piot, 1992). Similarly, cloning of a crylAc gene
from Btk into a Bt-aizawai, resulted in enhanced host spectrum. The original Bt-
aizawai showedgood activity against Spodopteraexigua. Therecombinant
product showed additional activity against a range of other species, including
Helicoverpa zea, Heliothis virescens, Trichoplusia ni and Plutella xylostelln
(Baum etal., 1990) (Figure 3.3).
In many instances, it has been found that introduced plasmid vectors
carrying isolated cry genes are unstable in Bt. Often, in the absence of selection
pressure, all or a portion of these plasmids are lost. The problem of plasmid
instability of introduced genes can be overcome byintegrating cloned cry genes
into the chromosomal DNA of the host cell. A cry1 C gene was introduced into
the chromosomal DNA of Bt-kurstaki tobroaden its hostrange(Glickand
Pasternak, 1998). The transformed Bt-kurstaki strain showed a six-fold increase
in its ability to kill Spodoptera exigua larvae.
Developmentofnovel Bt-based cloning vectors, such as Ecogen's
proprietary site-specific recombination (SSR) system, has made it possible to
construct improved Bt strains for use as microbial insecticide. This system could
selectively delete ancillary or foreign DNA elements (e.g. antibiotic resistance
genes) from recombinant cry plasmids after their introduction into a Bt host.
This has been used in the construction of a recombinant Cry3-overproducing
strain EG 7673 that became the active ingredient of the recombinant product
Raven@. Thestrain contains two coleopteran active genes, cry3A and cry3Bb, in
addition to crylAc. Apparently, the strain's overproduction of Cry3 protein

by cloning of Recombinant
Bt-ahawai U ~ I A Cgene product

I active
against

Spodoptera
exigua
I resulted in
ad& tional
activity

Helicowrpa zea,
Heliothis M'msoens,
Tkhoplusia ni and
Plutella xyiostella

Figure 3.3 Recombinant Bt-uizuwui cloned with crylAc gene from Bfk shows
an enhanced host spectrum.
84 Chapter 3

allows for more cost-effective use of the product for the control of Colorado
potato beetle (Leptinotarsa decemlineata) larvae (Baum et al., 1990).
Fusion of the coding portions of the active regions of two different
toxin genes is another way of generating a novel protein with extended toxicity.
In vivo recombination was used to produce hybrid genes constructed from
truncated c l y l A a and c ~ y I A cgenes, and two hybrid gene products produced in
this way acquired an entirely new activity against Spodoptera littoralis (Cramori
et al., 1991).
Thus, advances in recombinant DNA technology, which facilitated the
cloning of toxin genes and their expression in plants andvariousother
organisms, have increased the feasibility of using Bt for insect control. As a
result of the enormous potential that recombinant DNA technology provides for
advances in plant protection and other future insect control strategies based on
Bt, almost every major chemical company, and many emerging biotechnology
companies worldwide, are investing heavily in this area of research.

3.1 Bt Transgenic Microorganisms

The isolation of S-endotoxin has enabled scientists to characterize and clone the
genes encoding these molecules. Each &endotoxin is encoded by a single gene,
therefore it can be easily transferred to other Bts or to other microorganisms,
such as, bacteria, algae, fungi, virus etc. to create more stable/orcompatible
agents for the toxin delivery. Genetic engineering technology allows the use of
microorganisms that multiply on(epiphyte) or in (endophyte)plants to
continuously produce insecticidal protein at the site of feeding. Theprimary
rationale for using endophytic or epiphytic bacteria as hosts is to prolong the
persistence of Cry proteins in the field by using a host that can propagate itself
at the site of feeding and continue to produce crystal protein. By expressing
engineered cry genes in the transgenic hosts, their expression can be boosted to
higher levels than that in wild-type strains andhence can overcome broad-
spectrum resistance (Koziel et al., 1993b).

3.1. I Hosts for epiphytic delivery of Bt genes

In the early 1980s, Monsanto Company developeda recombinant, plant-


colonizing Pseudomonas for delivery of Bt genes, with the objective of
improving residual activity and efficacy of Bt proteins. Mycogen Corporation
hrther developed the concept to the first genetically engineeredbacterial
insecticide MVP@active against lepidopterous insects. They introduced cloned
Bt c t y l A c toxin gene into a microbial host, the root colonizing gram-negative
bacteria strains of Pseudomonas fluorescens. The recombinant P. fluorescens
Genetically Modified Bt Strains a5

cells that were subsequently killed by a proprietary chemicaltreatment


(Cellcap@technology), cross-linked the bacterial cell wall to yield a non-viable
encapsulated bacterium surrounding the crystal protein (Figure 3.4).
A number of commercial products have been developed based on this
process, including MVP@II,Mattch@and "Peril@ that are sprayed on the crop
like other Bts (Table 3.2). The apparent advantage of these strainsover
conventional Bts is that pseudomonad cell protects the Bt proteinfrom
environmental degradation, thus providing longer residual activity in the field.
Mycogen (1998) reported that MVP@IIprovided more persistent, long lasting
control of caterpillar pests than conventional Bt products. The exact mechanism
by which the cell protects the biotoxin is unknown, but the fixed cell wall
provides a mechanical barrier and may have the ability to impede a variety of
denaturing effects, including inactivation by sunlight (Gaertner et al., 1993). A
recombinant Bacillus megateriurn expressing a crylAa gene from Btk HD-1 also
reportedly persisted for more than 28d under field conditions, whereas Btk
disappeared within 4 days (Bora et al., 1994).
Stock et al. (1990) used cloning and conjugation technique to engineer
c j y l A c gene from Bt-kurstaki HD-1 into plant colonizing bacterium
Pseudomonas cepacia 526. The transconjugant P. cepacia cells produced only a
truncated (78 kDa) Cry protein, which protect axenically grown tobacco plants
from infestation by tobacco's hornworm.

Insecticidal Toxm producing Bacterlum Bacterla and


Heat kill
Iodine
crystal proteln gene 1s inserted reads the multiply bacterla stabilizlng
Into different genetic blueprint its walls and creatmg
bacterium and produces tiny capsules that
the toxm contam the t o m

Bacillus Pseudomonas
thunngiensis f/UOreXenS
bacterium bactenum

Figure 3.4 A Br cry gene is transplanted into another bacterium Pseudonzonas


fluorexens. These bacteria, when killed, form tiny capsules containmg
the toxin protein (Source: Mycogen Corporation).
86 Chapter 3

3.1.2 Hosts for endophytic delivery of Bt genes

An endophyte has been used to deliver &endotoxin to soil inhabiting insects.


The cry3A gene fiom Bt-tenebrionis was introduced into Rhizobium meliloti and
R. leguminosarum BV vicaea to protect alfalfa and pea crops from the feeding
damageoftwocoleopteran insects, larvaeofcloverrootcurculio (Sitona
hispidulus) and pea leaf weevil (Sitona lineatus) respectively (Bezdicek et al.,
1991) (Table 3.2).
Scientists at Crop Genetics International transformed the crylAc gene
from Bt-kurstaki HD-73 into the gram-positive endophytic
bacterium
Clavibacter xyli subsp. cynodontis (Cxc), for the control of European cornborer.
The transgenic Cxc, InCide@ infects the internal tissues of growing plants and
during growth produces Et toxin. As the corn borer larvae feed in the stem, the
insects ingest the endophyte and the Et (Lampel et al., 1994).

3.1.3 Hosts for Bt genes for mosquito control

One of the major factors limiting the duration of mosquito control followingthe
application ofBt-israelensis is the rapid sedimentation of spores andinsecticidal
crystals as the mostmosquitolarvaefeedatornearthewater surface. A
potential approach is to engineer microorganism living in the upper layer of
aquatic habitat to synthesize toxic protein. For example, cry4B toxin gene from
Bti was cloned into the broad host range plasmid pRK248 and expressed it in
Caulobacter crescentus CB 15, a motile ubiquitous bacteria. The recombinant
Caulobacter can provide the potential for prolonged control (Thanabalu et al.,
1992).
Mosquitocidal toxin genes have been also shuttled between Bacillus
sphaericus and Bt-israelensis to extend the host range to increase the toxicity
andpersistenceandtohelpdelay the appearanceof resistant mosquitoes.
Indeed, as the receptors for E. sphaericus and Bti toxins are different, placement
of Bti toxin genes into E. sphaericus recipient cells, would give benefits of both
worlds, the wider spectrum of Bti in a bacterium capable of surviving,persisting
andrecycling in the environment (Singer, 1990).When B. sphaericus strain
2362 was transformed withreplicative plasmids containing the cry1 1A gene, the
transformantsobtained
showed
various
segregational
orland structural
stabilities, some were moderatelytoxic to Aedes aegypti larvae (Trisrisook etal.,
1990). Similarly, recombinants expressing cry4B and cytlA genes,
independently or in combination, in E. sphaericus strain 2362, were more toxic
to Ae. aegyptithan the parentalstrain (Bar et al., 1991).
A novel approach for expressing Bti cryllA gene in E. sphaericus by
homologousrecombinationhasbeendescribed(Poncetet al., 1997).The
recombinant strain produced a large amount of Cry11A during the sporulation
Genetically Modified Bt Strains 87

Table 3.2 /It Transgenic


microorganisms

Transformed Trade Company/


Orgar?isnl Gene Source
Process larger
Nume Reference

Pseudomonas crylAc rDNA / MVP I I Mycogen Diamondback moth.


,Jluorescens (Btk) CellCap loopers. tobacco
budworm and cotton
bollworm

Bacillus CIyIAU Bora


rDNA et al..bollworms
Cotton
Megaterrurn (Btk HD-I) ( 1994)
(Strain KSI)

nucilllis cry1 IA rDNA Mosquito


Donovan larvae
Megaterium (Bti) et al.,( 1988)

Clavibacter cry IAc rDNA


InCide*
Lampel I<uropean
coni borer
xvli cynodonfis (Btk HD-73) et al.. ( 1994)
(MD 6PA)

RIIIzobiml cry3.4 rDNA BczdicekColoradopotato


melilofi (Bf- et al.. (1991) beetle. clover root
tenehrionls) lcaf pea curculio and
beetle

('aulobacter q-IB Shuttle 'l'hanabalu Mosquito larvae nt


Crescentus (Bti) vector et al., (1992)
or thenear water
C13 15 surface

Bacillus cr,vl /A Homologous Poncet et al.. Aedes aegypti and


sphaericus (Bti) Kecornbi- ( 1 997) Culex quinyui-
nation Jaciatus

B. sphaericus
cry-IB. Bar
rDNA et al., Aedes ueyyl,ti
(2362) cyr 1x1 (1991)
(Bfi)

sphaericus
LC. cry1 IA 'l'risrishook
rDNA Aedes aegypti
(2362) (Bti) et al.. (1990)

Pseudomonas cvylAc, rDNA./ Mattch


Mycogen
Broad spectrum of
jluorescens ccaterpillar
ylC CellCap pests,
(Bt-aizawai) such as beet arm).-
worm. diamondback
moth, corn earworm
etc.
88 Chapter 3

Table 3.2 (continued)

Trar~sfornred Trade Company/ Targel


orgalrisnl Getre Sorcrce Process
Name Reference
Pseudomonas
Btk Mycogcn
European
"Peril
rDNA/
corn
South
flrcorescens borer, CellCap
corn borer and fall
armyworm

Bt-kurstakr cryIAc ( 2 ) , rDNA


Raven
Ecogen
Lepidoptera
and
c93A and coleoptera
(EG insects
7673) on
cty3 Bb tomatoes potatoes,
and eggplant

Btk crylAa,and Fall


Ecogen
Lepinox
rDNA/ beet
crylAc (2), Protein
7826)
(EG
armyworm in sweet
cryZA, and Engineering
corn, other
turf and
C Q ~IF- I AC raw crops

Btk c r ) ~ l A c(3),
rDNN Crymax
spectrum
Ecogen
Broad
ctylC
activity
7841)
Protein
against
(EG
and
cry2A caterpillars
Engineering on fruits
and vegetables

* Not commercialized

process. The recombinant strain producedmorebinarytoxinthandidthe


parentalstrain.Synthesisof the Cry11Atoxinconferredtoxicity to the
recombinant strain against Aedes aegypti larvae, for which the parental strain
was not toxic. Interestingly, the level of larvicidalactivity of recombinant strain
against A n . stepltensi was as high as that of Bti. The toxicities of parental and
recombinant B. sphaericus strains against Culex quinquefasciatus were similar,
but the recombinant strain killed the larvae more rapidly.
The Bt-israelensis toxin gene cry1 I A hasalsobeenclonedinto B.
megaterium and the transformed bacterial cells were highly toxic to mosquito
larvae (Donovan et al., 1988). Similarly, the cly4A gene of Bt-isruelensis was
introduced into various unicellular cynobacteria with the intent of providing a
more accessible source of toxin for filter feeding dipteran larvae (Angsuthana-
sombat and Panyim, 1989) (Table3.2).
ModifiedGenetically Bt Strains 89

3.2 Protein
Engineering

Large scale screening of field-isolated Bt strains has been quite successful over
the years in identifyingnew Bt strains withincreased levels of insecticidal
activity as well as activities against a wide range of insect pests. Although new
crystal protein-encoding genes are being identified in these strains at an ever-
increasing rate, it is becoming apparent that highly active toxins, specific for
certain majorpest species, maynotbereadilyfound in nature. Today there
exists astrongmovementtowardproteindesignandengineering to help
overcomesome of the inadequaciesof the crystal toxins. Thisprotein
engineering approach could also be effectively used as a model system in strain
improvementanddevelopmentofhighly active andbroaderpest specificity
toxins (Powell etal., 1995).
Most of the current emphasis on engineering of the crystal protein is
aimed at characterizing their mode of action, that is, how the proteins exert their
effects at the molecular level. The possibilities for engineering novel Bt crystal
proteins with higher activities and wider host spectra are just beginning to be
explored.Ecogen(1998)reportedtechnology to improvethe insecticidal
capabilities of the Bt proteins through its work on the mechanisms involved in
the insecticidal activity. This technology, collectively referred to as mechanism
assisted insecticidal design, permits moving beyondnaturally occurring proteins
to identify new and commercially valuable proteins. Lepinox@, a product based
on recombinant DNA andproteinengineeringtechnology to create novel
insecticidal proteins, has been commercialized. It has increased activity against
several species of armyworms, while maintaining the favorable environmental
and safety aspectsofa naturally occurring Bt. Similarly, anotherproduct
Crymax@, that containsacombinationof three proteintoxins CrylAc,a
modified CrylC and Cry2A, provides a high level of potency and spectrum of
activity against caterpillar pests (Table 3.2).
Rajamohanal.
et
(1996)
reported that by
using site-directed
mutagenesistechmques, there is a significant enhancement in toxin-receptor
contactandsubsequentlyimprovedbinding affinity andpotencyof CrylAb
toxin. Wu and Aronson(1 992) observedthat a mutation in helix a5 of domain I
of Cry1 Ac, caused a two-fold increase in toxicity against M. sexta, that was
found to be correlated withthe rate of irreversible binding.

4 Insect-Tolerant Transgenic Crop Plants


The use ofBt for crop protection has evolvedrapidly, from thedirect application
of the sporesand crystal toxinsof this bacterium as biopesticide, to the
developmentoftransgenic plants expressingclonedtoxingenes.Genetic
engineering has created transgenic variety of many crop plants that express Bt-
Chapter 3

toxins (Huang et al., 1999: Schuler et al., 1998). Although the potential exists to
transfer most of the insecticidal principles found in diverse organisms, the
current work has focused on transfer of insecticidal genes of bacterial origins, in
particular from Bacillus thuringiensis to various crop plants. The transgenic
plants armed with Bt-toxins are defended against some of the most notorious
pests, which reduce the need for insecticidal sprays.
The transgenic plants provide season long protection, independent of
weather conditions, effective control of burrowing insects and others that feed at
sites difficult to reach with sprays and control at all the stages of insect
development. The important feature of such a system is that only insects eating
crops are exposed to the toxins. Because Btis not toxic to arthropod natural
enemies, opportunities for biological control are enhanced and the secondary
pest outbreaks often caused by conventional pesticides are avoided.Such
transgenic cropsprovide farmers an attractive alternative over traditional
application of chemical pesticides and a means of controlling serious insect
pests, not easily controlled by current chemical pesticides. The use of transgenic
plants could also overcome some of the stability problems associated with
conventional Bt applications. Thus, the new technology could yield enormous
benefits for food production and environment quality worldwide.
Bt strains contain great diversity of 6-endotoxins encoding genes and
have proved to be a remarkable source of insecticide principles to be used in
transgenic plants. Bt-insecticidal proteins have been successfully expressed in a
wide range of transgenic plants, including cotton, corn, tomatoes, potatoes, rice
and soybean.Their expression have been achieved at levels that provide
economic control of lepidopterous pests, including cotton bollworms, tobacco
budworm, pink bollworm and the European cornborer. This has introduced a
new dimension, in the utility of Bt-toxins asacrop protectant in transgenic
plants. Severalcompanies including Monsanto and Novartishave received
registration for transgenic cotton and corn seeds by the EPA. The first
generation of insect resistant plants, such as transgenic corn, cotton and potatoes
were grown on a large scale in the United States during 1996.

4.1 Dicotyledonous Plants

The Bt genes crylAa, clylAb or crylAc isolated from lepidopteran active Btk,
have been introduced into tobacco, tomato or potato. The toxin genes were
introduced by Agrobacterium-mediated transformation (Figure 3.5).
Agrobecterium tumafaciens is a plant pathogen causing tumorous crowngalls on
infected dicotyledonous plants. Agrobacterium-mediated gene transfer has been
a well established method to introduce foreign genes into plants, albeit restricted
to dicotyledonous plants and their expression conferred somedegree of
protection against tobacco pests (Manriuca sexta), tomato pests (Heliothis
Genetically Modified Bt Strains 91

Agrobactenum plasmld
contalnlng the Insect 'Iasmid wntalning
control proteln gene
Bacillus tbunnglensm

Agrobactenum transfers the


Insect control protem gene
Into plant cell chromosome

Agrobacterium
turnafaciens

The Improved cell is


cultured and grown
mtoa whole plant

Figure 3.5 Cotton plant resistantto insect pests. Each cell contains the
lnsectlcldal protein gene, providingthe cotton plant with the ability to
ward off attack by caterpillar pests (Courtesy Monsanto Company).

virescens and Helicoverpu zeu) and potato pests (Phthorimueqoperculellu).


Perlak et al. (1990) reported on the performance of transgenic cotton plants (var.
Coker 312) containing crylAb or crylAc with effective control of cotton pests
such as tobacco budworm (Heliothis virescens), pink bollworm (Pectinophora
gossypiellu) and moderate population levels of cotton bollworm (H. zea).
The development of cotton cultivars with transgenes usually involved
two distinct phases. The first phase involved selection of transformation events
that express the &endotoxin protein at the desired level, without any major
negative effects on agronomic and fiber properties. The second phase involved
hybridization of the selected transformed plant with elite germ plasm, followed
by selection and evaluation of progeny to determine the expression of the 6-
endotoxin and the agronomic and fiber properties of the selected lines (Jenkins,
1999). A modified 6-endotoxin-encoding gene c1y3A, isolated from coleopteran
active Bt-tenebrionis was transformed into potato plants conferring protection
from damage by the Colorado potato beetle (Leptinotarsa decemlineatu) at high
levels of field infestations (Adang et al., 1993; Perlak et al., 1993). These initial
accomplishments were restricted to dicotyledonous plants.
92 Chapter 3

Bt cotton has been released commercially in the USA, Australia and


South Africa. Bt potatohasbeenapproved for commercialization in USA,
Canada and Japan (Schuler et al., 1998). The commercial transgenic lines of
cotton and potato are given in Table 3.3.

4.2 Monocotyledonous Plants

Transformation of monocotyledonous plants, is based on technology of direct


gene transfer, where DNA transformation is mediated by non-biological means.
Polyethylene glycol-mediated transformation, electroporation or microprojectile
bombardment has been developed as possible approaches. The microprojectile
bombardment looks especially promising, primarily because it allows the direct
delivery of DNA into a wide range of plant cells, obviating the regeneration of
plants from transferred protoplasts. Koziel et al. (1993a) reported the expression
of Bt-genes in monocotyledonous plants by transforming elite cultivars of maize
with a truncatedcrylAb gene.
Ciba Seeds (now Novartis Seeds) and Mycogen Seeds introduced the
first Bt corn hybrids in 1996. These transgenic corn plants provided excellent
protection against European corn borer (Ostrinia nubikalis) even at a very high
insect larvae pressure. Several seed
companieshave
incorporated this
technology into their best inbred lines. Seed companies select elite hybrids for
the Bt transformation in order to retain important agronomic qualities for yield,
harvestability and disease resistance. Various commercialized corn lines contain
improved expression of one of three Cry protein genes active against European
corn borer, i.e. CrylAb, CrylAc or Cry9C. Most ofthe Bt corn hybrids, produce
onlythe CrylAb protein; afewproduce the CrylAcprotein or the Cry9C
protein.

Table 3.3 Commercializedtransgenic Et cottonandpotato

Gene Plants Trade Name Company Target

Cotton crylAc Bollgard


Monsanto budworm
Bollworm and

Potato cry3A Monsanto Colorado


potato
beetle
NewLeaf NatureMark
cry3A+
Leaf NewLeaf Monsanto/
Colorado
beetle
potato
Potato Virus resistance Plus NatureMark and potato leafrollvirus
gene
Genetically ModifiedBt Strains 93

Bt Cry protein gene

t
(modified for improved expression)
Genetic consists of A promoter
gene
xpression)
toxin (controls
package
A marker gene, such as genes
for herbicide resistance
or antibiotic resistance
(Identifies successful transformations)

Figure 3.6 Geneticpackagecomponents

Thegeneticpackage inserted into corn consists of three primary


components: a Br Cry protein gene, a promoter gene that controls where, when
andhowmuchof the toxin is expressedandamarkergene that allows
identification of successful transformations (Figure 3.6). Current examples of
markers include genesfor herbicide-tolerance or antibiotic-resistance.
Successful transformations, called "events," vary in the components of
the genetic package and where this DNA is inserted into the corn DNA. The
insertion site may affect Bt production and could affect other plant functions
(Figure 3.7). Thelevel of European corn borer (ECB) control against late-season
ECB infestations differs between Bt events. Under heavy ECB pressure, events
B T l l andMON810provideahigher level of controlthanevent 176. The
differencecould be explainedby the fact that event176hybridsproduce Bt
protein only in green tissues and pollen, whereas B T l l and MON810 events
produce it throughout the plant. Because some hatching larvae initially colonize
ears to feed on silks and developing kernels, these larvae may surviveon

Genetic package
components v"ying in v
Successful
and insertion siteinto
transformations
the corn DNA
I (Bt events)

functions
plant
other
Bt protein production
and
iof
Transgenic corn

Figure 3.7 Construction of transgenlc Bt corn


94 Chapter 3

event 176and may tunnel later into stalks andearshanks(Ostlieand


Hutchinson, 1997).
The transgenic Bt corn has been commercially released in the USA,
Canada, Argentina, Japan and the European community (Schuler et al., 1998).
The various commercialized Bt corn lines are given in Table 3.4.
A modified crylAb genehasbeen inserted intoriceusing micro-
projectile bombardment technique to enhance crylAb gene expression. The level
of expression of the modified gene in transgenic rice was0.05% of total soluble
leafprotein. The plantswere significantly resistant to two lepidopteran rice
pests, leaf folder (Cnaphafocrosismedinafis)and stemborer (Chzlo suppressah)
(Fujimoto et. al., 1993).

4.3 SecondGenerationInsectResistantPlants

The second generations of insect resistant plants are under development. These
include both Bt and non-Bt proteins with novel modes of action and different
spectra of activity against insect pests. For example, proteins such as cholesterol
oxidases
and
members of VIPs (vegetative insecticidal
proteins)
family
represent the second generation of insecticidal trans-genes that will complement
the novel Bt &endotoxins. A maize plant has been transformed which expresses

Table 3.4 Commercialized transgenic Bt corn

ene Name Trade

YieldGard cty/Ab MONS I O Monsanto European corn borer, corn


earworm, fall armyworm
YieldGard crylAb BTI 1 Novartis European corn borer,
Seeds southwestern corn borer, corn
ear worm and fall army worm
Knockout ctylAb 176 Novartis European corn borer
Seeds
Attribute ctyIAb BTll Novartis European corn borer and
Seeds southwestern corn borer
Naturecard ctylAb 176 Mycogen European corn borer
Bt-Xtra ctylAc DBT418 Dekalb European corn borer
StarLink oy9C CBH351 AgrEvo European corn borer and
black cutworm
ModifiedGenetically Bt Strains 95

a VIP and a Bt &endotoxin, by genetically engineering the plant to contain and


expressallthegenes necessary (Warren et al., 1999). Thesecancontrol
economicallyimportant insect pests such asnorthernandwesterncornroot-
worm and bollweevil, which are not effectively controlled by any known Bt 6-
endotoxin. In an effort to enhance the Bt b-endotoxin gene expression in second
generation plants, the genes with sequence modificatiofi and/or new promoters
includingtissue-specificpromotersarebeing used (Estruschetal.,1997).
Biotechcompaniesarealsoworkingtodevelop Bt crops with stackedor
pyramided genes that can produce two different Bt toxins with two different
modes of action This will reduce the likelihood of pest resistance and will lead
to arelaxation in refuge requirements and otherregulatoryfactors(Renner,
1999).

5 Concluding Remarks
The recent elucidation of the X-ray crystal structures of Bt proteins opens new
possibilities for protein engineering and design of Bt insecticides. Determination
of the mode of action at the molecular level and the genetic basis for insect
specificity should enable recombinantDNA technology to be used to expand the
insect host range of Bt as well as increase its toxicity against insects. In the
future, it is easytoimaginethatcrystalproteinvariants,engineeredfor
improved toxicity, yieldor stability by in vitro mutagenesis, may be used to
steadilyimprove the performance of Bt-based insecticides. In addition, the
construction of hybridtoxinswith improved insecticidal activity will provide
novel active ingredients suitable for commercial exploitation. It is anticipated
that engineeredformsof toxin proteinsshowingimprovedpotencyor yield,
regardlessoftheirhost, will make Bt bioinsecticidesamoreattractive and
practical alternative to synthetic chemical control agents.
Advances in understanding of proteinstructure and functionare
allowingscientistsusingprotein-engineeringtechnologies,tobegin to design
chimericproteins.Thesechimerasareconstructed by piecingtogether at the
gene level discrete functional parts of the protein, called domains that can be
swapped to change or increase their normal species activity spectrum.The long-
term goal of protein engineeringis construction of modular smart proteinsthat
will target specific pests and,like Bt, not harm beneficial animals.
The use of transgenic plants expressing Bt &endotoxin provide a level
of insect control not seen since the introduction of chemical insecticides decades
ago and have the potential to greatly reduce the environmental and health costs
associated with the use of conventional insecticides. Advances in tissue culture
have beencombined with improvementsintransformationtechnology to
increase transformation efficiencies particularly in case of corn and cotton. It is
96 Chapter 3

anticipatedthatsecondgenerationtransgenicplantsproducinginsecticidal
proteins with either differentmodes of action or different targets or both options
consisting of multiple genes into the same plant would reduce the possibility of
resistance development.

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24. Koziel, M. G., G. L. Beland,C.Bowman, N.B. Carrozi, R. Crenshaw, L.
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M. Wrightand S. V. Evola, 1993a.Fieldperformance of elitetransgenic
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http://www.mes.umn.edu/
I3ocuments/D/C/DC7055.html
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G. Reed, G. Biever and D. A. Fischhoff, 1993. Genetically improved potatoes:
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This Page Intentionally Left Blank
Formulation of Bacterial Insecticides

1 Introduction
The purpose of formulation of a microbial system is to produce a product that
optimizestheeffective and economical use of atoxin,withinaparticular
environment. Bacillusthuringiensis subsp. israelensis(Bti) formulation,a
mosquito biolarvicide, for example, is used in an aquatic environment. On the
other hand, Bacillus thuringiensis subsp. kurstaki (Btk) formulation, a product
for control of agricultural insect pests, is applied through foliar application. In
order to be effective, both of these products should be easily accessible to their
respective target larvae and should be reasonably stable under field conditions.
Thus, even though the nature of insecticidal crystal proteins (ICPs) and spores
aresimilar in both cases, the final producthastobeapplied to different
environments, with specific requisites for accessibility and stability.
There have been a number of recent reviews on the formulation and
application of bacterial and viral insecticides (Burges et al., 1998; Devisetty et
al., 1998; Shieh, 1998; and Cibulsky et al., 1993). Various aspects and attributes
of ingestible microbial formulations targeting for maximum effectiveness with
specific referenceto Bt and Bs, are discussed in this chapter.

101
102 Chapter 4

2 Characteristics of Microbial Insecticide Formulations


In general, formulation processes and specifications of microbial systems are
quite similar to that ofchemical pesticides. However,microbialssuch as
bacteria or viruses differ from chemicalpesticides in several aspects as follows:

2.1 Particulate
Characteristics

Insect pathogens are particulate materials composed of high molecular weight


proteins. Unlike synthetic pesticide molecules,thetoxin particulates are
insoluble in aqueousandorganic solvents. In fact, any effort todissolve
particulate toxinscauses loss of toxicity. These are living entities withan
organized cellular structure entrapped in agenerallyhydrophobicmembrane.
This limits the options available for formulating microbial pesticides to wettable
powders,suspensionconcentratesandwater dispersible granules. Surface
bondedgranularsand briquette formulationsare also possible for specific
applications. However the insolublenatureof the active ingredientdoesnot
permitformulations
such as emulsifiable concentrates
and
water
based
concentrated emulsions.

2.2 Ingestion of Particulate Toxin by Target Larvae

Microbial agents suchas bacteria, viruses etc. must be ingested by target pests in
order to be toxic. Thus, palatability ofbiopesticideformulationsassumes
significance in termsof ingestible particle size, as well as role offeeding
stimulants in enhancing ingestionrate.

2.3 Stability
Characteristics

Microbial systems are generally more sensitive to the surrounding environment


thanmost chemicals.Thus, the stability of microbialsystemsformulations
requires special care for maintaining biological activity. Some of the factors that
affect the stability and bioactivity of the formulationsincludepH,choiceof
surfactants and high temperature. The hgher pH level often results in malodor
formation and bacterial growth. Stabilization of Bt in aqueous vehicles is much
morecomplex as several interacting factors such as hydrolysis,microbial
degradation, oxidation, proteinaggregationcanadversely affect the stability.
Similarly, use ofanionic surfactants inan aqueousformulationmayoften
denature the protein structure of toxin. Prolonged exposure to high temperature
Formulation of Bacterial
Insecticides 103

during the production process and subsequent storage may also result in loss of
insecticidal activity.

2.4 BioassayasAnalyticalMode

A formulation active ingredient is analyzed by bioassay against species ofpests.


Alternativechemicalmethods,such as direct measurementofproteinor
immunoassayof&endotoxin in Bt formulationsare valid only if extensive
correlation studies are established between the bioactivity andchemical
quantification.

3 Formulation Attributes Having Impact on Effi-


cacy of Bacterial Insecticides
Insect pests develop in a multitude of ecological niches. Various factors need to
be taken into account to developa bacterial or viral formulation,including
biology, susceptibility and feeding behavior oftarget pest(s), and information on
type and nature oftheir breeding habitat. Some of the formulations canbe tailor-
made for a single site, pest or use-pattern. Different attributes that may lead to
optimization of formulation efficacyare discussed here.

3.1 Particle Size Influences Placement and Ingestibility of Toxins

The particle size of the suspended formulations can exert considerable influence
on application and efficacy. It is known that the settling rate of a formulation
corresponds directly to its mean particle size. Thus, a microbial formulation for
mosquitocontrol that enables flotation of toxic constituentsnear the surface
over a sustained period of time would enhance residual toxicity against surface
feeding mosquito larvae.
It has been observed that a flowable formulation with a smaller mean
particle size, settled more slowly than wettable powders and provided prolonged
control. Khetan et al. (1996) studied the relative efficacies of two commercial
formulationsof Bti in laboratoryand field against Anopheline larvae. The
formulationscomparedwereawettablepowder(Bactoculicide)ofahigher
potency (5400 IU/mg) and an aqueous suspension (Aquabac@) (1200 IU/mg).
Application of an equal amount of both the formulations in the laboratory test
resulted in higher mortality for powder formulation consistent with its relative
higher potency. On the other hand, aqueous suspension formulation was found
more effective in the field studies, when equal amounts ofthe two formulations
104 Chapter 4

Table 4.1 Comparativeefficacyofpowderandaqueoussuspension


formulations ofBti in the field studies (Khetan et al., 1996)

Fortnulatiorrs o j Potency"
Reduction in Anopheline Larvae Populatiorl (96)
Bti IUIme
Days 1 3 7 14

Wettable Powder
5400 100 100 79
(Bactoculicide@)
Aqueous suspension
1200 100 100 84 47
(Aquabac@)
(a) IU = International Units

wereapplied(Table 4.1). Investigations revealed that a fine particle sized


aqueous suspension formulation of Bti out performed a higher strength powder
formulation for control of aquatic surface feeding mosquito larvae.
The ingestion of particles by mosquito larvae is also governed mainly
by size. Generally,mosquitolarvaeingested particles between>0.5pmand
<50pm in diameter. In an X-ray diffraction analysis for particles ingested and
retained by larvae ofAedes triseriatus, it has been observedthat 1'' and 2"dinstar
larvae ingested particles of <2 pm, 3rd instars ingested mostly particles in 2-
10pm range, whereas, 4" instars ingested mainly particles in the range of 10-
25pm. Only a small proportion of smallerparticles (<2pm) was ingested by 3rd
and 4' instar larvae (Table 4.2).
Skovmand et al. (1997) studied the influence of particle size on toxicity
by conducting bioassays on 2nd and 4' instar larvae of Ae. aegypti employing
severalcommercial Bt-isruelensis formulations.Theirfindingsrevealed that
smaller the median particle size, steeper the slope of the concentration-mortality
curve, i.e. LCsovalue becomes lower.

Table 4.2 Particles ingested and retained by larvae of Aedes triseriatus


as determined by X-ray diffractlon analysis

Aedes triserlatus
larvae stape Particle size Ingested

1St and 2"d instar <2 pm


3rdinstar 2{ -A1 0 p m proportion
of small
smaller particles (<2 pm)
41hinstar I O - 25 pm (mainly)
was
also
ingested }
Formulation of Bacterial
Insecticides 105

3.2 Feeding Stimulants Enhance Palatability and Anti-Feedants Reduce


Efficacy of Bt Products

3.2. I Feedingstimulants as adjuvants

The Bt-based bioinsecticides are gut toxins and must be ingested by the target
larvae in order to effect mortality. Their toxic effects can be enhanced, if the
feeding rate of the pests could be increased. To this end, use of a feeding
stimulant or phago-stimulant would increase efficacy by increasing the
concentration and rate of toxins ingested.
Lepidopteran larvae are reported to have a reduced feeding rate on
foliage or artificial diet treated with Btk, or they avoid feeding in favorof
untreated foliage. The feeding behaviour oftobacco budworm (Heliothis
virescens) on artificial diet treated with Btk formulation was reported to result in
feedingavoidance(Gould et a1.,1991) (Figure 4.1). Similar observation of
reduced feeding on Btk treated feed was reported with gypsymoth (Lymantrio
dispar) larvae. However, addition of nutrient based feeding stimulants
(consisting of proteins, lipids and carbohydrates) in the formulation increased
the initial dose ingested by the larvae, killing them and thus preventing recovery
and feeding resumption (Farrar and Ridgway, 1995) (Figure 4.2).
Similarly, in mosquito larvae, Rashed and Mulla (1 989) observed that
dried yeast, wheat flour, fish meal and dried blood were ingested at significantly
faster rates than inert particles such as kaolin, talc, chalk and charcoal. Yeast
contains a numberof phago-stimulants, which stimulate feeding activity of
mosquito larvae and it increases ingestion rate in several species of mosquitoes
too.

on foliage
Lepidopteran larvae
(Tobacco budwrrn,
spruce budwonn and Bt-kurstak,
European corn borer)

In favor of
untreatedfoliage Feedingrate

Figure 4.1 Lepidopteranlarvaereducefeeding on foliageorartificialdiet


treatcd with Bfk.
106 Chapter 4

Gypsy moth
(Lymantria dispar )
on treatment
with - Bt-kurstaki
formulation
reduced Feeding

-
ad& tion of
a feeding
stimulant to
Bt-kurstaki increased ingestion of killing
formulation initial dose them
preventing

recovery and
feeding resumption

Figure 4.2 Feedingstimulants improvepalatability o f B t formulation.

Bartlet et al. (1990) investigated the palatability of a starch matrix based


granular formulation of Bt-berliner by European corn borer larvae and found the
larvae seldom fed these. On the other hand, the granule acceptability improved
considerably, with addition of a mixture containing lipid, sugar and protein.
Coax, a commercially available phago-stimulant (Table 4.3) was most effective
in improving acceptability, increasing it 320-fold. Further, when Coax was
added to granules, the amount of Bt could be reduced by 75%, without reducing
mortality, indicating that larvae fed long enough to be killed. A similar response
under field conditions wasconfirmed by McGuire et al. (1990). In a comparative
study of the effect of commercially available nutrient based phago-stimulants on
various lepidopterous insect larvae, it wasobserved that Pheast, containing
substantial amounts of carbohydrates (39%) and protein (44%), produced best
response for all insect species (Farrar and Ridgway, 1994). A list of
commercially available phago-stimulants, their sources and major constituents
are given in Table 4.3.

3.2.2 Presence of anti-feedmt with Btproducts

Basedow and Peters (1997) reported the effect of individualformulations of


azadirachtin (Neem Azal@) and Bt-tenebrionis (Novodor@) on control of
Colorado potato beetle (Leptinotarsa decelineata) in potato. They also studied
the effect of their combination by application on consecutive days. The results
are summarized in Table 4.4.
Formulation of Bacterial
Insecticides 107

Table 4.3 Commerciallyavailablephago-stimulantsprayadjuvants


(Adapted from Farrar and Ridgway, 1994).

Maior Constituents (x)


Product - . I

Carbohydrate
Protein

sno,
Agrisense,Pheast CA 39.0 43.8
CCT, Coax Litchficld Park, AZ 50.5 32.8
Gusto Atchem-North
Amerlca,
Philadelphia,
34.1 57.7
PA
Consume'Fermone,Phoenix,AZ
Chemicals,
Frenso,
Custom
Entice CA 54.8 35.3
Industries,
Loveland
Mo-Bait
Greeley, CO 2.8 95.0
(a) Identical product to Gusto

Azadirachtin is an effective antifeedantand insect growth-regulating


agent. The results indicate that combination of azadirachtin and Bt-tenebrionis
asa single sprayeach reduced their individual effectiveness significantly. It
appears that azadirachtin has an antagonistic effecton Bt-tenebrionis due to
reduction or near stoppage after initial feeding by the larvae. It is apparent that
insects distinguish antifeedants as they distinguish feeding stimulants.

Table 4.4 Effect of Azadirachtm and Bt-tenebrionis formulations oncontrol


of Colorado potato beetle.

Control of Colorado Feeding damage Yield (potato)


Fortnulation
potato beetle (%
' ) C%) (Kg120plants)

Rt-tenebrronis 11.20 20 ( 1 19%)


55.4
(Novodor 31/ha)

Azadirachtm
71
(129%) 12.14 7
(Neem Azal 0.5%)
Combination (5%)
(Neem Azal 0.1%+ 5 10.5744 ( 1 12%)
Novodor 1.5 Iha)

Untreated 0 63 9.45 ( 1 00%)


108 Chapter 4

3.3 Non-Toxic and Mildly Toxic Additives: Potentiation


of the
Activity ofBt-Toxins

3.3.1
Proteaseinhibitors The
additionof
protease inhibitors to
protect
the proteinaceous toxin is reported to potentiate Btk and Bti activities many fold.
MacIntosh et al.(1990) reported the addition of extremely lowlevels of protease
inhibitors enhanced the insecticidal activity of Btk by 2-20 fold against a variety
oflepidopterans.A similar effect wasseen in Bti formulation tested in
combination with soybean protease inhibitor against Aedes aegvpti, potentiating
activity 3 fold. It is postulated that protease inhibitors may inhibit the
degradation of membrane-boundreceptors, thus increasing thehalf-lives and the
ability to bind Bt proteins.

3.3.2
Allelo-chemicals Plants
produce
allelo-chemicals to resist insect
attack. These chemicals could also increase the efficacy of biological control
agents. Tannins,amajorconstituentof Texus sp. causes larval mortality.
Addition of tannicacid, an expensive commercial source oftannin, was tested to
increase the efficacy of sub-lethal concentration ofBt. Supplementation with 25-
500ppmtannicacidyielded55-75% mortalities of Trichoplusiani. In
comparision, Bt alone produced only a 10% mortality (Gibson et al., 1995).

3.3.3 Non-toxic chemicals Effectiveness


of Bt-berliner on addition of
several non-toxic chemicals has been reported againstSpodoptera (Salama et al.,
1985).Inorganic salts (eg.carbonatesofsodium.potassiumandcalcium,
sulphates of magnesium and zinc), boric acid, lipid emulsifying agent such as
Tween 60 and protein solublizing agents have been found to enhance efficacy
significantly. These findings have been corroborated in field trials. It was found
that addition of 0.075% potassium carbonate, zinc sulphate or calcium carbonate
to the microbialsuspension,lead to a greatly enhancedefficacyof Btk
formulation(DiPel)against S. littoralis onsoybean,both in termsof larval
population reduction and cropyield increase (Morris et al. (1995).

3.3.4
Effect of addition of EDTA Lin
andTabashnik(1997)reported
interesting observations of EDTA additionto Btk. They observed that feeding of
EDTA by itself to diamondback moth larvae did not result in mortality, but Btk
along with EDTA reducedLC50 by 2-3 fold in the resistant larvae (both neonates
and 31d instars). In case of susceptible larvae, combination with EDTA did not
lead to reduction of LCs0 Btk of towards 31d instars, although in case of neonates,
there was a 5-fold reduction in LC5oof Btk (Figure 4.3). It is clear that addition
of EDTAsignificantly increased the toxicity of Btk.
Formulation of Bacterial Insecticides 109

Resistant
Diamondback
moth -w Susceptible
larvae larvae xylostella)
(Plutella

feeding
feeding
neonates 3rd
instars
Significantly
reduced LCso Do not kill Reduced LCSO No effect
( 2-3 fold ) larvae ( 5-fold )

Figure 4.3 Effect o f addition o f EDTA on the efficacy of B f k formulation.

3.4 Protein
Enhancin
Synergises Bt

Enhancin is a 104 KDa enzymatic protein which has been found to synergise
activity of Bt as well as nuclear polyhedrosis viruses(NPVs) in noctuid moths at
levels of 50 picogramsper insect (Menn, 1996). The interior lining of the
insects intestine, the peritrophic matrix (made mostly of chitin and proteins) is
an important component in the insects immune system against certain microbial
attack(biologicalcontrolagents).Enhancin, when consumed by the insect,
binds to a major mucinous protein of peritrophic matrix and destroys the matrix
structure. Thus, apparently the passage of biocontrol agents (Bt, baculoviruses)
is facilitated through this relatively impermeable matrix unimpeded, resulting in
the rapid death of the insect.

3.5 Effect of Adjuvants to Enhance Efficacy

The ability of sprayed microbial formulation to remain intact on a leaf surface,


not withstanding adverse
environmental conditions, is prerequisite
a to
achieving the optimum activity inherentwith the microbial system. Factors such
as photo-degradation, volatility, spray drift and rain fall, all effectively reduce
the potential for the pest control agent to reach the target. Bt does not exhibit
significantactivitybeyond 3-4 dayson the targetfoliage,whenexposed to
sunlight, moisture and leaf exudates. A number of approacheshave been tried to
protect Bt and thereby enhance the persistence on the foliage. Modifications of
current Bt formulations via the addition of adjuvants have been used as one of
the approaches.
110 Chapter 4

3.5.1 Rain
fastnessenhancement The addition of commercial wetting,
sticking and spreading agents is recommended when sprays are applied to waxy
surfaces such as cabbage and broccoli or when rain fastness is important. An oil-
based formulation is reportedly less susceptible to wash off by rain, than
aqueous-based formulations, presumably due to better adhesion of the droplets
(Devisetty et al., 1998). Earlier, these authors had reported the findings of W.
Mclane (Cibulsky et al., 1993), that adjuvant Rhoplen B60A improved the
rain-fastness of Bt on oak seedlings, by demonstrating 43% improved mortality
of gypsymoth larvae as compared to Bt formulation without the adjuvant.
Narayanan and Fanniello (1996) reported improved rain fastness by addition of
Agrimax 3 (Intl Speciality Products) composed of pyrrlidone-based solvents,
surfactants and water insoluble polymers micro-dispersed in aqueous medium.

3.5.2 Photoprotectionenhancernent Sunlight inactivation of Bt hasbeen


demonstrated to be partly responsible for its short persistence in the field.
Pusztai et al. (1 987) conducted photo-stability studies with pure &endotoxin
crystals from Bt strains HD-1 and HD-73. Their results demonstrated that the
300-380 nm range of the solar spectrum was largely responsible for loss of
toxicity. Cohen et al. (1991) reported photoprotection of the toxic component by
adsorption of cationic chromophores, suchas acriflavin, methyl green and
Rhodamine B to Bt. It was reported that acriflavin gave the best protection. A
mechanism involving the energy transfer from the excited tryptophan moieties
to the chromophore molecules has been suggested. The insect pigment melanin
has been found remarkably effective at concentration as low as 0.0003% (Pate1
et al., 1996). Tamez-Guerra et al. (1996) investigated photo-stability of spray
dried gelatinized starch-based granule formulations of Bt. Theyfound that
addition of lactic or citric acid with starch/flour materials provided increased
protection against solar radiation. This was attributed to matrix-like granule
formation on spray drying, where the active ingredient is distributed throughout
the granule (Le., active ingredient is on the outside as well as within the
granule).

3.5.3 Flow Property ofBtformulations good


A flow property of Bt
formulations is a requirement for good spray-droplet deposition.A viscosity
range of 700 m. pas. or lower has been suggested to give better atomization
efficiency (Devisetty et al., 1998). Use of thickening agents can alsocontrol
drift and evaporationof aerial and ground sprays of Bt.

Ignoffo et al. (1976) used a commercial adjuvant to reduce evaporation,


increase stability in sunlight and increase larval feeding on foliage treated with
formulation. Thus depending on specific requirement, a wetting agent, oil,
Formulation of Bacterial
Insecticides 111

polymerizingagent,spreader, penetrant, sunlightprotectant,humactantand


thickner can beselected as adjuvantsto increase coverage and effectiveness.

4 Commonly Used Formulations of Bt


Bt fermentation slurry
concentrate
is
either
dried to technical
powder
concentrateorformulatedasstabilizedaqueoussuspension. Bt spraydried
technical powders are processed for particle size control and utilized in both
liquid (aqueous and non-aqueous suspensions)and wettable powder (WP), water
dispersiblegranules(WG) andimpregnated coatedgranularformulations.
Wettable
powders and
water dispersible
granular
formulations
are
predominantly used for pest control on vegetable, agronomic and fruit crops.
Non-aqueous and aqueous suspension formulations are widely used for forestry
and raw crops suchas cotton and soybeans (Devisetty et al., 1998).
Specific applications also justify surface bonded granular formulations,
for example, for penetration of dense foliage, for mosquito larvae control or as
briquettes
for
hand
application in an aquatic environment where spray
application is not practical. The relative merits of various formulation types used
for Bt-based pest control products aregiven as follows:

(a)
Wettable powderWettable
powdersare
dry,
free
flowingpowders,
containing a high concentration of active ingredient(s). They are formulated to
facilitate mixing with water into a final spray. The quality of water dispersible
powder is judged by the rapidity of wetting when mixed with water, and its
suspensibility in water when mixed in practical dilutions for field application.
Fine particle sizeimprovessuspensibility.Sizereductionisachieved via air
milling or hammer milling of the product, to achieve a particle size range of 10-
20 pm. Typical examples include DiPel WP (Abbott), Agree (Therm0 Trilogy)
and Cutlass WP (Ecogen).

(b)Suspensionconcentrates(Flowables)Thesearepreformedsuspensions
of well-dispersed micronized active ingredients either in water or in oil phase.
These are processed by wet milling (Dynomill) of microbial agents along with
adjuvants to achieve the particles in 1-2 pm sizes. These formulations have the
advantage of being relatively simple to recover and formulate. However, such
formulations may also show limited storage stability, perhaps due to higher rate
ofproton transfer acrossthe microbial cellularmembraneinanaqueous
medium. Some of the typical examples are Florbac FC (Novo Nordisk), Mattch
and MVP I1 (Mycogen) and VectoBac FC (Abbott).
112 Chapter 4

(c)Water dispersible granules(WG)These are formedfrom all technical


powders, i.e. agglomerated with binders. The preferred process of formulatingis
by fluidized bed granulation of fine slurry. The formulation must wet instantly
when put in water and possess good dispersibility characteristics. Commercial
WG formulationsare stable productswithexcellenthandlingandmixing
properties. Some of the typical examples include Javelin WG (Thenno Trilogy)
and XenTari WG (Abbott). Ecogen has marketed its genetically modified Bt-
productsCrymaxandLepinox also as WG formulations.HoweverCrymax
WDG is prepared by extrusion ofthe cell paste, as against spray drying, givingit
desirable handling properties (Baum etal., 1999).

(d) GranularsGranularformulationsofamicrobialsystemfrequentlyuse
an inert carrier with a specific size. The microbial agentis bonded to the carrier
with various types of sticking agents. These formulations are useful for aquatic
larvae control to treat larval habitats under vegetative cover. Examples include
DiPel 10G (Abbott) and "Peril (Mycogen).

(e)BriquettesA briquette is a solid blockformulationwithadiameter


ranging from 2 to 6 cms. Briquettes are generally formed by mixing the active
ingredientswith light density inert granules and
binding
agents.
These
formulations allow timely release of individual granules and are useful for the
spreadingof
microbials into water.
Briquettes
are
convenient for hand
applications into aquaticenvironmentswherespray
applications are not
available or not functional. A typical example is Bactimos briquette (Summit
Chemical, Baltimore).

5 Improved Bt Formulations
With advances in culture, production, formulation and application technologies,
Bt formulations have undergonesignificant improvements from the conventional
low-potencyformulations.Productionofhigh-potencyfermentedtechnical
materials
has
contributed in goodmeasure to this development. The
development of improved formulations also provided a means to increase the
effectiveness of entomopathogens. Pioneering work has been done in forestry
for the control of
gypsy
moth (Lymantria dispar), spruce budworm
(C~~ol.istoneurafumifernna), and other forest caterpillars. Optimized droplet size
and distribution ina forest canopyfromvarious aircraft, combinedwith
undiluted, high potency formulations have resulted in greatly improved control
oflepidopterous forest pests. Particularly, oil-basedemulsifiablesuspensions
Formulation of Bacterial
Insecticides 113

and encapsulation techniques have helped the pest control agent reach the target
and remain there by withstandingthe environmental occurrences.

5.1 Oil-Based Enrulifiable Suspensions (ES)

The development of ULV Bt formulation of highpotencyasstableoil


emulsifiable suspension (ES) formulation has provided more consistent insect
control than dry wettable powder formulations or low potency aqueous-based
formulations. The oil-based formulationcan be applied directly, without dilution
with water andprovidessuperiorspraycoverage on targetfoliage.The
formulationpotencyrangingfrom 8.45 Billion InternationalUnits(BIU)/l to
32.82 BIU/l also results in improved efficacy due to presence of concentrated
dose of Bt in each spray droplet. These formulations are compatible in both
water and oil systems and can be tank mixed with many oil based chemical
pesticide formulations and non volatile diluents such as cotton seed oil. It has
been reported that expansion of Bt from original vegetable crop applications to
field crops suchas cotton and soybean andforest insect control programs inhard
wood and coniferous has been facilitated with these developments (Cibulsky et
al., 1993). These formulations are also reported to maintain excellent biostability
during storage (Devisetty et al., 1998). Some ofthe examples of emulsifiable oil
suspension concentrates includeDiPel ES (Abbott), Condor XL (Ecogen) and
Delfin ULV (Therm0 Trilogy).
The Bt usage has increased in combination with non-evaporating spray
oilsandotheradditivesincotton,corn and soybeaninsectcontrol.Thus, Bt
provided effective control of cotton insects such as tobacco budworm (Heliothis
virescens) and the bollworm (Helicoverpa zea). (Figure 4.4). Similarly, aerial

Low volume
Soybean looper
High potency superior [Pseudoplusia
aerial Bt ES - control -t
includens)
application formulation

J
Forest insects
eradication programs
(Gypsy mothand
spruce budworm)
1
1
Cotton pests, e.g.
tobacco budworm
and bollworm
1 comparcd
to

Lambdacyhalothrin
(Karate)

Figure 4.4 High-potency low volumeapplicationof Bf providcseffective


control of insects In agrlculture and forestry.
114 Chapter 4

application of Bt ES formulation for control of soybean insect control,


specifically for soybean looper (Pseudoplusia includens) have produced
equivalent or superior control to synthetic pyrethroid lambdacyhalothrin
(Karate@- Zeneca)
Some of the other improved Bt formulations that are available include
high-potency aqueous flowable concentrates, non-aqueous emulsifiable
suspension formulations and non-emulsifiable oilsuspension formulations.
These are highly suitable for low volume (4.7 to 18.7 Vha) and ultra-low volume
(< 4.7 l/ha) applications on agronomic crops and for forestry use. Some
commercially available formulations are listed in Table 4.5.

5.2 Encapsulated
Formulations

5.2.I Starch
matrix
based
encapsulation: Dunkal and Shasha (1988)
reported encapsulation of Bt in a granular starch matrix that improved the
residual activity of B. thuringiensis for the European cornborer (Ostrinia
nubilalis). Shasha and Dunkel (1989) patented a process for encapsulating
biocontrol agents such as pathogenic bacteria and viruses in a protective starch
matrix without the use of chemical cross linking agents. The biocontrol agent is
blended into a dispersion of pre-gelatinized starch, which is then subjected to
conditions suitable for retrogradation. The dispersions can be formulated either
for recovery of drygranules or as sprayable liquids. McGuire and Shasha (1995)
also reported a sprayable starch encapsulation incorporating starch and sucrose
into a water dispersible formulation by employing a different process.This
starch-based formulation was found effective in maintaining residual activity of
Bt on cabbage.

Table 4.5 Commercial Btk LV /ULV formulations

Trade
Mamrfacturer
Fornlulation Potency
name

DiPel ES Emulsifiable oil Labs.


Abbott 16.91 BIU/L
suspension
Foray 48 B Aqueous flowable Nordisk
Novo 12.86 BIU/L
concentrate
Thuricide 48 LV Aqueous
flowable
Thermo
Trilogy 12.70 BIU/L
concentrate
DiPel 12L Non-emulsifiable
Abbott
oil Labs.
suspension concentrate
(a) BIU = Billion International Units
Formulation of Bacterial
Insecticides 115

5.2.2 Cellcap@encapsulation system: MycogenCorporation(SanDiego,


California)producedencapsulatedformulations of increasedresidual,high
potency bioinsecticides based on their Cellcap@ encapsulation system. In this
process, the single Bt-toxins are introduced in a microbial host Pseudomonas
jluorescens. The recombinant P. jluorescens cells are subsequently killed by a
proprietary chemical treatment that cross-links the bacterial cell wall to yield a
non-viable encapsulated bacterium surrounding the crystal protein (see figure
3.3). An example is MVP@II, a product based on CrylAc &endotoxin of Bt-
kurstaki. In a comparative evaluation, Mycogen (1998) reported that MVP II@
providedmore persistent, long lasting control of caterpillar pests
than
conventional Bt products such as DiPel@ and Javelin@ (Figure 4.5). The exact
mechanism by which the cell protectsthe biotoxin is unknown, but the fixed cell
wall provides a mechanical barrier and may have the ability to impede a variety
of denaturing effects, including inactivation by sunlight (Gaertner et al., 1993).

5.2.3 MatricapTMencapsulatiorz: Levy et al.(1996)reportedaMatricap


granular formulation that encapsulated biolarvicides such as Bt-israelensis, for
slow release through coating-regulated controlled delivery system from floating
or submerged matrices. These formulations have been found useful in targeting
the feeding zones of larvae of Anopheles, Aedes and Culex mosquitoes in fresh
and brackish water.

100
(2.34 LiUHa)
80
60
40
20
0
t (1.46 kglha)
I
0 4 7
Days after treatment

Figure 4.5 Foliar


persistence
of MVP@ I1 formulated Bt versus
conventional formulatedBts (Adapted from Mycogen, 1998)
116 Chapter 4

6 Target-Specific
Tailor-Made Formulations of
Bacterial Larvicides
Anopheles mosquitoes commonly breed i n stagnant rain water pools, open
storage tanks and other water holding containers. The larvae are adapted to
collect particulates from air-water interface. An effective microbial formulation
could make use of the feeding behaviour of Anophelines by suspending the toxin
only in the feeding horizon, i.e. at or near the water surface. Other desirable
features of the formulation could include an ability to penetrate barriers, such as
naturalfilms,vegetation etc. and remain floating for a long duration without
degradation.
The standard water diluted sprayable formulations such as wettable
powders and suspension concentrates disperse in the whole water body and soon
settle to the bottom. Thus, significant quantities of toxin particles will be outside
their feeding zone. Treatment with granules, pallets and briquettes are not
appropriate in this situation. The settling feature of the formulations render them
ineffective against larvae hatching out of eggs laid soon after treatment. Thus,
there is a need for specific formulation(s) for this particular situation. It would
appear that a floating type of formulation may increase activity by remaining in
the feeding zone much longer.
Ramdas and Khetan (1990) reported a tailor-made self-spreading oil
formulation of microbial insecticides that displayed many of the desired
properties. The product, a stabilized suspension of the micronized toxicant in an
oil phase containing lyophilic surfactants dispersed in an alcohol was found
suitable for ultra low volume (ULV) application. The droplets of the product on
contact with water spread spontaneously with a great force (spreading pressure
>60 dynedcm) into a micro-reticulum (figure 4.6a) that soon began expanding
(figure 4.6b), ultimately breaking down to evenly.distributed discrete micro-
globules (figure 4 . 6 ~ )The
. micro-globules were of optimal dimensions (-5 p n )
for rapid tapping andingestion by the mosquito larvae (figure 4.6d). The
formulation was found to be effective at a fractional concentration as compared
to an aqueous flowable formulation.
Formulation of Bacterial Insecticides 117

Figure 4.6 Tailor-madeself-spreading oil formulation of Bti keeps thetoxin


particles floatingin the larval feeding zone
118 Chapter 4

7 Efficient Delivery is Equivalent to Effectiveness at


Low Dose
Application technology, timing, rate of application, drop size, density (number
ofdropletsper cm) andweatherconditionscan effect controlof insect
population. Method of application has been found to have significant effect on
the efficacy of Bt. Observations on field application of Bt products for control of
diamondback moth (Plutella xylostella) employing a knapsack, a drop nozzle
andan electrostatic sprayerhavebeenreported(Perez et al., 1995).The
electrostatic technique showed significantly better performance as compared to
other two applicationtechniques. Electrostatic applicationpossibly results in
greater deposition of electrically charged droplets on the plant than that obtained
with conventional hydraulic nozzles.
Earlier, Law (1983)reportedapplicationof Btk to control T. ni in
broccoli. Application through an electrostatic sprayer required only 1/2 - 113 the
recommended dose per hectare compared with a conventionalsprayer. The high
efficacyobtainedwiththe electrostatic applicationdeviceatlow rates will
reduce the number of treatments per season.

8 Concluding Remarks
Microbial formulations areat the very end of the downstream processing part of
the production of microbial larvicides. Effective microbial formulation depends
upona variety ofcomplexareasincludingbiologyandfeedingbehavior of
insect pests, ecological requisites of species, individualcontrol agents, their
effectivenessand their physico-chemical properties. Rigorousdemandsare
placedon features such as effective dose delivery, palatability, particle size,
placementand persistence, stability overasustainedperiod,and cost. A
judicioususeofformulationtechnology, e.g., oil suspensionemulsions in
combinationwithapplicationtechnology,such as lowvolumeapplicationor
electrostatic application can be effective in enhancing activity and prolonging
the useful life of microbials as pest control agents. Finally, for the optimum use
of the specificity of microbials tailor-made formulations are the key rather than
multi-purpose standard formulations.

References

1. Bartlet, R. S., M. R. McGuireand D. A. Black, 1990. Feedingstimulants for


the European corn borer (Lepidoptera: Pyralidae): Additives to a starch based
formulation for E t , Environ. Entonlo/., 19,182-1 89.
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Insecticides 119

2. Burges, H. D., andK. A. Jones, 1998. Formulation of bacteria, viruses and


protozoa to control insects,In Formulation of Microbial Pesticides: Beneficial
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potato
beetle
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berliner
against
four
lepidopterous
insect
pests
nutrient
by based
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insecticidal
biotoxin
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15. Khetan, S. K., M. A. Ansari, P. K. Mittal and A. K. Gupta, 1996. Laboratory
and field bioeficacy studieson Bacillus thururgiensis var. israelensis (Serotype
H-14) against Culex and Anopheline larvae, Unpublished Results.
16. Law, S. E., 1983.Electrostaticpesticidespraying:concepts,andpractice,
IEEE (Inst. Electr. Electron. Eng.)Trans. Ind. Appl., IA-19, No. 2.
17. Levy, R., M. Nicols andW.R. Opp, 1996. New Matricap pesticide delivery
systems, Proc. Int I Symp. Control Rel. Bioact. Mater., Kyoto, Japan, Control
Rel. SOC..23.
18. Lin, Y-B and B. E. Tabashnik, 1997. Synergism of Bacillus thuringiensis by
EDTA in susceptible and resistant larvae of diamondback moth (Lepidoptera ;
Plutellidae),J.Ecorr. Entonrol., 90, 287-292.
19. Machtosh, S. C., G. M. Kishore, F. D. Perlak, P. G. Marrone, T. B. Stone, S.
R. Sims and R. L. Fuchs,1990.Potentiationof Bt insecticidalactivityby
serine protease inhibitors, J. Agric. Food Chetn.,38, 1 145-52.
20. McGuire, M. R., B. S. Shasha, C. E. Eastman and H. Oloumi-Sadeghi, 1996.
Effect of starch and flour based sprayable formulations on rainfastness and
solar stability of Bacillus thuringiensis, J. Ecorr. Elrtotnol., 89, 863-869.
21. McCuire, M.R.andB. S. Shasha,1995.Starchencapsulationofmicrobial
pesticides, in Biorational Pest Control agents: Formulationand Delivery, F. R.
Halland W. J. Barry,eds.,AmericanChemicalSociety,Washington,DC,
pp.229-237.
22. McGuire, M. R., B. S. Shasha, L. C. Lewis, R. J. Bartlet and K. Kinney, 1990.
J. ECOII.E1~to~trol., 83, 2207-2210.
23. Menn, J. J., 1996. Biopesticides: Has their time come?, Environ. Sci. Health,
B3 1(3), 383-389.
24. Molloy,D., S. P.Wright, B. Kaplan, J. GerardiandP.Peterson,1994.
Laboratory evaluation of commercial formulations of Bacillus tkuringiensis
var. israelerrsis againstmosquito andblackfly larvae, J. Agric. Entoarol. 1,
161-168.
25. Morris, 0. N., V. ConverseandP.Kanagaratnam,1995.Chemicaladditive
effectsoftheefficacyof Bt-berliner srcbsp.
kurstakl agalnst Metnestra
conjgurata (Lepidoptera: Noctuidae), J. Econ. E~rtonrol..88, 8 15-824.
26. Mycogen Tipsheet - MVP@II, 1998.http://www.mycogen.com/graphic/pest
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27. Narayanan, K. S. and R. M. Fanniello, 1996. Superior multipurpose adjuvant
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eds., American Society for Testing of Materials.
28. Patel, K. R., J. A. Wyman, K. A. Patel and B. J. Burden, 1996. A mutant of
Bacillus thuringiensis produclngadark brown pigmentwithincreased UV
resistance and insecticidal activity, J. Invertebr. Pathol., 67, 120-124.
29. Perez, C. J., A. M.sheltonand R. C. Derksen,1995.Effectofapplication
technology and Bt subspecies
on
management of Bt-kurstaki resistant
diamondback moth (Leprdopteru : Plutellidae), J. Econ. Entornol., 88, 1 1 13-
1 119.
Formulation of Bacterial
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30. Pusztai, M., P. Fast, H. Kaplan and P. R. Carey, 1987. The effect of sunlight
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Conway, eds., 2,39.
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This Page Intentionally Left Blank
Insect Resistance to Bf Toxins

1 Introduction
Resistance is a genetically based decrease in susceptibility of a population to an
insecticide. As resistance entails changes in allele frequencies in a population,
bydefinition, it isanevolutionaryphenomenon.Insectsare known fortheir
ability to developresistance to certain insecticides rapidly.Resistanceoccurs
particularly when insecticidesare used repeatedlyand at highconcentrations
leading to intensiveselectionpressureon insect populations.Morethan 500
speciesofinsects andmites are known to have developedresistanceto
insecticides and miticides (Georghiou and Lagunes-Tejeda, 1991).
To document development of resistance, one must show that repeated
treatments with an insecticide have caused a significant increase in the amount
of insecticiderequiredto kill a certain proportion of population(e.g. the
concentration that kills 50%, LCs0) or a significant decrease in the percentage
mortality causedbya fixed amount of insecticide.Thedegree of resistance
observed in an insect population is typically expressed as the resistance ratio, i.e.
number of LCso-resistantinsects / number of LCso-sensitive insects.
Laboratory colonies of more than 15 different insect pests have been
demonstrated to develop resistance to Bt proteins, including Indian meal moth,
tobacco budworm, beet armyworm, pink bollworm and Colorado potato beetle.
Moreover, the diamondbackmoth,aworld-widepest ofcolecrops, has
123
124 Chapter 5

developedhighlevelsofresistance to Br insecticide In fieldpopulations in


several countries.
Insects couldpossiblyachieveresistance to Bf toxins by different
mechanismsrangingfrom the pointofprotoxiningestion to theinsertion of
toxin in the membrane. The factors affecting the bindingof toxin to the receptor
would result in selective resistance to one particular Bt toxin. On the other hand,
those steps utilised by all the toxins viz. proteolysis of protoxins, conformational
alterations and membrane insertion may lead to cross-resistance to multiple Bt
toxins. Reduced binding ofBt toxin to the brush border membraneof the midgut
epithelium was identified as a primary mechanism of resistance to Indian meal
moth and diamondbackmoth.
Several authors have reviewed different aspects of insect resistance to
Bt toxins and its management (Schnepf et al., 1998; Bauer, 1995; Tabashnik,
1994; Marrone and Macintosh, 1993; and McGaughey and Whalton, 1992). In
this chapter, an overview of resistance to Bt toxins has been presented. The
discussionincludes the synergisticinteractionsbetweenvariousCryproteins
andCryproteins and spores.Currentviews and approaches on resistance
management have also been coveredhere.

2 Resistance to Bt toxins in Lepidoptera


More than twodecadesoffrequentapplications of Bt-based formulationsin
many parts of the world, have resulted in the development to Bt spore-crystal
toxins complex resistant populations of lepidopteran caterpillars. It is known
that toxicity of Bt-toxins relates to their ability to bind to receptors in the larval
mid-gut and a single insect may have different receptors for different Bt-toxins.
There has been evolution of insect resistance to insecticidal proteins and it has
been found that resistance is toxin-specific. The mechanism of resistance relates
to change in binding characteristics of Bt-ICP against Lepidoptera, dueto either
areduction in the concentration of microvillarmembranereceptors,reduced
toxin affinity for the receptors, or an implied failure of the toxin to insert into the
microvillar membrane afterbinding. A decrease in the affinity between the toxin
and target receptors is frequentlyassociated with amutation on thetarget
receptor (Figure 5.1).
McGaughey (1985)
observed that Indianmeal
moth (Plodin
irrrerpunctella) populations from grain storage binsthat had been treated for 1 to
5 months with a Bt-kurstaki formulation had a small but significant increase in
LC50s relative to populations in untreated bins. Further, he demonstratedin
laboratory experiments, that after 15 generations of selection, insects from the
treated colonyshowedLCs0snearly 100-fold greaterthanthoseshown by
control experiments. VanRie et al. (1990) demonstratedthat the resistance
Insect Resistance to Bt Toxins 125

Development
of resistance

relates

It Of

t
due to

Bt-ICP against Mutation on


Lepidoptera target receptor

Figure 5.1 Mechanlsm of resistance development in Leprdoptera against


Bt through decrease in affinity.

correlated with a significant reduction in affinity of the membrane receptor for


the CrylAb protein, one of the toxins present in the Bt-kurstaki formulation
(Dipel@) used for selection (Figure 5.2). Evidence was also found that in
resistant P. interpunctella larvae, in addition to changes in binding, there may be
decrease in cleavage of the full-length Bt protein to its toxic form due to a loss
of protease function (Oppert et al., 1997).
The Indian meal moth (Plodiainterpunctella), as discussed, has
probably evolved low levels of resistance in stored graintreated with Bt
(McGaughey, 1985). Resistance in another stored grain pest almondmoth
(Cadra cautella) has been reported only from laboratory studies (McGaughey
and Beeman, 1989). Similarly, resistance developmentintobaccobudworm
(Heliothis virescens) (Stone et al., 1989, Gould et al., 1992 and Lee et al., 1995)
and the Colorado potato beetle (Leptinotarsadecerulineata) (Whalonetal.,
1993) has been also reported from laboratory selectionexperiments.The
resistance trait proved to be recessive. When selection was removedbefore
resistance became fixed, resistance levels decreased. However, the very
possibility of resistance development in the field populations is of great concern
because of their economic significance.
Although, at least eight species have adapted to resist Bt S-endotoxins,
only one species, diamondback moth (Plutella xylostella) has actually developed
significant levels of resistance under field conditions. The mechanism of
resistance in diamondback moth population that had developed resistance in the
field was demonstrated to be also due to change in ICP membrane receptor
(Ferre et al.1991). It has been found that resistant populations of diamondback
126 Chapter 5

Plodia
interpunctella
(Susceptible)
wmw
......
.. .....
...
.. ......
....
.. ...... Midgut
lumen

Plodia
interpunctella
(Resistant)

crylAb CrylAb DefectiveCryiAb


receptor receptor
CrylC pq CrylC
receptor

Figure 5.2 Mechanism of resistancetoICPs in the Indian meal moth (Plodiu


interpunctellu). The brush border membrane of the midgut of wild-
type(susceptible)larvaecarriesreceptorsfor CrylAb and CrylC
toxins that make them sensitive to these toxins. Resistance to Cryl Ab
in Plodiu (resistant) is due to defective receptor for the toxin. The
CrylAb resistant larvae are still sensitive to CrylC, which binds to
anotherreceptor. No cross-resistanceisobserved (Lambert and
Peferoen, 1992).

moth to CrylA toxins from Bt-kurstaki, showed cross resistance to CrylF, but
not to CrylB or CrylC toxins (Tabashnik et al. 1996). It has also been reported
thatdiamondback moth withpopulationstreatedwithBt-aizawaidisplayed
resistanceto CrylC (Liu et al., 1996).OnHPLCanalysis ofcommercial
formulations of Bt-kurstaki and Bt-aizawai, was
it apparent that CrylAb was the
most abundant Cryl protein in both, while CrylC was present in Bt-aizawai
alone. The CrylC resistantdiamondback moth populations were found resistant
to CrylAb, but thiswas lower than in a CrylC susceptible colony that had been
selected by Bt-kurstaki (Figure 5.3). The toxins in the cross-resistance group
have significant amino acid sequence similarity in domainII, a region believed
to be important for receptor binding in many systems (see Chapter 1).

2.1 Bt Spore-Crystal
Interactions

For many lepidopteran insect pests, such as the beet armyworm (Spodoptera
exigua), the Btsporespresentonbioinsecticideformulationalsocontribute
al., 1995). The synergistic effectof spores has
substantially to toxicity (Moar et
also been reported for gypsy moth (Lymantria dispa) (DuBois and Dean, 1995).
Insect Resistance toBt Toxins 127

treated Diamondback moth treated


Bt-kurstaki 4 with t Bt-aizawai
with population

J
became became
cross- resistant to
resistant to

CrylCrylA F cry1c
(Btaizawar)

1
toxins partidy
remaincd cross-
susceptible resishnt to

Cryl B or ClylAb
CrylC toxins

Figure 5.3 Resistance toBt-toxins in diamondbackmoth.

A study of laboratory colony of Spodoptera littoralis treated with Bt-


spore crystal preparations of CrylC toxin, developeda resistant population
(>500 fold resistance) in 14 generations. This population exhibited partial cross-
resistance to CrylD, CrylE, CrylAb toxins and to parental strain Bt-aizawai.
However, their susceptibility to CrylF was unchanged(Muller-Cohnet al.,
1996) (Figure 5.4).

14 generations
(lab. selection)

CrylD,
CrylEand partial Resistant
strain cross-
* Bt-aizawai
CrylAb toxins
P
cross-resistance ( > 50 -fold ) resistane

I
t
cross-resistance
no

Cryl F

Figure 5.4 Cry 1 C-resistant S. littoralis shows partialcross-resistance


to othcr Cry-toxins.
128 Chapter 5

Tang et al.(1996)studiedtoxicity of Bt spore and singlecrystal


protein, to resistant population of diamondback moth. The diamondback moth
larvae tested, showed a high level of field-evolved resistance toBtk spore and all
the three CrylA protoxins, but still susceptible to CrylB, CrylC and CrylD.
When Bt-kurstaki spore and single cystal protoxin preparation (1 :1) was fed to
susceptible larval of diamondbackmoth, spores synergised the activity of CrylA
( 5 to 8-fold) and CrylC (1.7 fold), respectively. On the other hand, when this
combination was fed to resistant larvae, spores failed to synergise activity of all
three CrylA protoxins, but synergised the activity of CrylC (5.3- fold), (Figure
5.5). In a bid to determine possible mechanisms of resistance, binding studies
with CrylAb, CrylB and CrylC were performed. The resistant larvae showed a
dramatically reduced binding of Cry1Ab as compared with that in susceptible
larvae, butno difference in bindingof CrylB or CrylC.
Liu et al. (1 998) extended the study of interactions between spores and
single cystal Bt-toxins to interactions of spores and combination of toxins that
occur in crystals from Bt-kurstaki and Bt-aizawai. In leaf residue bio-assays on
diamondback moth larvae, they reported synergistic interactions on addition of
Bt-kurstaki spores to Bt-kurstaki crystals. A reduction in LCso of Bt-kurstaki
crystalsagainstsusceptiblelarvae(10-fold)andresistantlarvae(45-fold)
respectively, was observed.On the other hand, sporesof Bt-aizawai had little or
no effect on toxicity of crystals of Bt-aizawai.
The interactions between spore and single toxins revealed synergism
between Bt-kurstaki spores and Cry2Aa against susceptible diamondback moth
larvae. Preparations of Bt-kurstaki spores alone caused significant mortality of
susceptiblelarvae,while Cry2Aa alonedid not causesignificantmortality.
Synergism also occurred between Bt-aizawai spores and CrylC toxin against
both susceptible and resistant larvae. Although, again Bt-aizawai spores alone
did not cause significant mortalityin susceptible or resistant larvae.

Resistant
fed to Susceptible
diamond back fed to Bfk spore and ___t diamond back
protoxin
larvae moth (1:l)

I
motharvae

synergised

Activity of
Activity of ActM ty of C y l A (5-8 fOM)
C v l C (5.3-fold) ClylA
clylc (1.7-fold)

Figure 5.5 Synergistic


activity of Cry proteins.
Resistance
Insect to Bt Toxins 129

Thisspore effect on the insecticidal activity of Bt maybe due to


septicemia, the ability of the sporeto germinate within the insect midgut to
penetrate the disrupted midgut epithelium, and to enter and proliferate within the
hoemcoel. For many lepidopteran insect pests, it is therefore desirable that Bt
bioinsecticide formulations contain a mixture of spores and crystals to achieve
maximum efficacy. An implication of this study on Bt-toxin expressing
transgenic plants and transgenic bacteria is that absence of spores may
accelerate evolution of pest resistance. However, such a possibility would need
to be experimentally evaluated.

2.2 Bt Cry-Cry Interactions

In a study, the toxicity of toxins CrylAa,CrylAb and CrylAc wasfound


against gypsy moth (Lymantria dispar) by force-feeding bio-assay(Leeet
a1.,1996). CrylAa exhibited higher toxicity than toxins CrylAb and CrylAc.
When bioassay was conducted with mixtures of CrylAa and CrylAb, CrylAb
and CrylAc, and CrylAa and CrylAc, a synergistic activity was observed with
the mixture of CrylAa and Cryl Ac. On the other hand, a mixture of CrylAa and
CrylAb exhibited an antagonistic effect (Figure 5.6).

CtylAa,
Gypsy moth Force-fed
(Lymantria dispar)
CrylAa
bio-assay * CrylAc
and

Bio-assayed C r y l h and exhibited ~ Synergistic


against activity Cry 1Ac
mixtures
c r y l ~ and
b exhibited Additive
CrylAc activity

CrylAa and exhibited ~

CrylAb activity

Figure 5.6 Force-feedbio-assay of gypsy moth withindividualandmixtures


of Cryl A proteins.
130 Chapter 5

3 Synergistic Interactions of Bf-israelensis Toxins


and Effect on Resistance Development
Despite several years of field usage, no significant resistance has been detected
in field populations of mosquitoes towards Bt-israelensis even in areas where
they havebeentreatedintensively.Attemptsatlaboratoryselectionhave
produced only low levels of resistance in Culex quinquefasciatus leading to the
hypothesis that the heterogeneous mixtures of toxins present in Bt-israelensis
constituteaneffectivedefenceagainst the developmentofresistance. Bt-
ismelensis crystalscontainfour major &endotoxins,namelyCry4A,Cry4B,
Cryl 1A(earlier known asCryIVD) and CytlA. Crickmoreetal. (1995)
determined their relativeactivitiesthrough bio-assay against Aedesaegypti
larvae.Bio-assay of mixturesof the individual toxins revealedanumber of
synergistic interactions. Poncet et al. (1995) evaluated synergistic interactions
among the Cry4A,Cry4B and Cryl 1Atoxiccomponents of Bt-israelensis
crystals against Aedes. aegypti, Anopheles stephensi and Culex pipiens. It was
found that Cry4A and both Cry4B and Cryl 1A gave synergistic effect while
Cry4B and Cryl 1A showed a simple additive effect (Figure 5.7). Other crystal
toxins presumably contribute to full toxicity of wild type Bti crystals. This also
explains, as to whynative crystal is considerably more toxic than any of the
individual toxins.
Wirth,
Georghiouand
Federici
(1997)studied
the
synergistic
interactionsof CytlA with Cry toxins (Cry4A,Cry4Band Cryl 1A) of Bt-
ismelensis. They observed that CytlA, in addition to being toxic, synergises the
toxicity of Cry proteins (Table 5.1). CytlA is a highly hydrophobic endotoxin
with an affinity for Bt-israelensis Cry toxins, but shares no sequence homology
with the Cry proteins and appears to have a different mode of action. Whereas,
Cry proteins initiallybind to glycoproteins onthe microvillar membrane,

Et-israelensis crystal proteins


( Cry4A, Cry4B and Cryl 1A )
twocomonent mixtures
bioassayed

Cry4A with both


Cly4B and Cry1 1A
Cry4B and Cry1 1A

Figure 5.7 Toxic effect of mixture of Bt-isruelensis Cry proteins.


Insect Resistance to Bt Toxins 131

Table 5.1 Synerglsmbetween CytlA andCrytoxins of Bt-israelensis tested


against larvae of C. q~trnquefasciatus(Wirth et al., 1997)

Cry 1 I A + CytlA 0.32 1 3.0 0.978


Cry4A + Cry4B + Cyt 1 A 0.0501 0.36 1 7.2
Cry4A + Cry4B + Cry1 1 A + CytlA 0.0141 0.0380 2.7

primary affinity ofCytl A isfor the lipid component of themembrane,


specifically,for unsaturated fatty acids in the lipid portion of microvillar
membrane. It is believed that CytlA may be the key protein accounting for the
inability of mosquitoes to quickly developresistance to Bt-israelensis. They also
tested the combination of sub-lethal quantities of CytlA with Cry proteins ofBt-
isrnelensis and found that it suppressed or markedly reduced high levels of Cry
resistance in the mosquito Culex quinquefasciatus.
Georghiou and Wirth (1997) also studied the influence of Bt-israelensis
toxins as a model system to determinethe speed and magnitude of evolution of
resistance in themosquito Culex quinquifasciatus duringselectionfor28
consecutivegenerations with single or multiple toxins.Resistancebecame
evident with single toxinCryl 1A, when by 28th generation (FZ8)resistance ratio
(RR) at LC95 was >913. Resistance was found to evolve slowly and at low level
with two toxins Cry4A and Cry4B, as at Fz5, RR was found to be >122. It was
observed to be lower still with three toxins Cry4A, Cry4B and Cryl 1A (RR=91
at Fzg). It was found to be remarkably low with all the four toxins (RR = 3.2)
(Table 5.2).

Table 5.2 Development of resistance in C. quinquefasciatus with Individualand


mixture of Bt-israelensis toxins (Adapted from Georghiou and Wirth,
1997)

Bt-israelensis C. quinquifasciatus Resistance ratio


toxins Peneration at LC,,

Cry 11A F?8 > 913


Cry 4A + Cry 4B F2s > 122

Cry4A+Cry4B+CryII~ F28 91
Cry 4A + Cry4B + Cry 1 I A + C y t l ~ F2s 3.2
132 Chapter 5

Thestudy provided evidence for two hypotheses regarding Bt


resistance: (a) that combinations of Bt Cry proteins are better than Cry proteins
used alone, and (b) that using a protein like CytA, with a different mode of
action, in combination with Cry proteins, may delay the development of
resistance.

4 Insect Resistance to B. sphaericus


Bacillus sphaericus inclusions are made of equimolar amounts of proteins of 52
and 42 kDa which act asa binary toxin that has apotential for resistance
development. It is reported that Culex quinquefasciatus develops resistance to B.
sphaericus at various degrees. Treatments with preparations of B. sphaericus at
high dosagesand high frequency has given rise to resistance development
ranging from 30-40 fold to 10,000 fold, depending on the intensity of selection
pressure. It has beenobserved that the resistant strains of larvae of Culex
quinquefusciatus ingested B. sphaericus particulates at a significantly lower rate
than did larvae of susceptible strains. Since ingestion of toxin particulate is a
necessity for expression of toxicity, it has been suggested that a change in favour
of a lower ingestion rate may provide an initial mechanism for emergence of
resistance (Rodcharoen and Mulla, 1995).
B. sphaericus binary toxin bind to larval midgut cells in Culex species
with high specificity and high affinity, leading to high susceptibility of these
species. The mechanism of resistance to B. sphaericus toxin as well as Bt-toxins
seems to depend on similar non-functionality of membrane midgut receptors
(Nielsen-Leroux etal., 1995). However, it hasbeenobserved that mosquito
populations resistant to B. sphaericus remain susceptible to Bt-ismelensis
toxins. In binding experiments, no competition was observed between B.
sphaericus toxin and Bt-israelensis ICP toxins, indicating that different
receptors are involved.

5 Resistance Management of Bt Toxins


Bt-toxins are used as an environmentally benign foliar spray and are among a
handfd of microbial biocontrol agents available to conventional growers, who
want safe, efficient pest control tools and to organic farmers, who are concerned
about loosing one of their most reliable biocontrol tools. The challenge is to
develop and implement strategies that provide adequate short-term insect control
while delaying insect resistance to Bt-toxins. This will ensure that the long-term
economic and consumer benefits are preserved and value of this
environmentally useful insecticide is not jeopardised.
Resistance
Insect to Bt Toxins 133

5.1 Resistance
Monitoring

Establishment of baseline susceptibility in field population of target pests toward


insecticidal protein, precisely characterised in terms of its nature and amount of
&endotoxins, would be the first step.Monitoringisalsonecessarytolearn
whethera field controlfailure resulted fromresistanceorotherfactorsthat
mightinhibitexpression of the Bt protein. Thus,centralto the operation of
resistance management program is an effective monitoring program to detect as
early as possible, shifts in pest susceptibility that could be abated in the initial
stagesofresistanceproblem.Resistancemanagementprogramsarebest
implementedbefore the pestbecomesresistant.Detectingresistancemaybe
possiblebeforecontrol failures occur, if monitoringtechniquesaresensitive
enough to providecompletediscriminationbetweenresistantandsusceptible
individuals. A monitoring strategy for early detection of Lepidoptera resistance
to Bt-ICPs has been reported by employing larval growth inhibition assay using
sub-lethal CrylAc doseon Heliothisvirescens and Helicoverpazea. This
approach is claimed to be more sensitive than dose-response mortality assays. It
allowed visual discrimination of resistant from susceptible phenotypes (Sims et
al., 1996).

5.2 General
Strategies

Resistance management strategies have been proposed as a means to decrease


the rateat which resistanceevolves and hence to keep the frequency of
resistance genes sufficiently low for insect control. The development of strategy
relies heavily on theoretical assumptions and on computer models simulating
insect population growth under various conditions. Tabashnik (1994) outlined
the strategies in the context of P. xylostella - B. thuringiensis system. These
included,(a) the use of mixtures of toxins withdifferentmechanisms, (b)
synergists to increase toxicity, (c) mosaic application to resort to
time
alternations rather than space alternations, (d) rotation of toxins to reduce the
frequency of resistant individuals, (e) ultra-high dosesof toxin that kill resistant
heterozygotes and homozygotes and (0 refiges tofacilitatesurvival of
susceptible individuals.
Perez et al. (1997) tested, some of these strategies that were essentially
basedonthe results oflaboratoryinvestigations,onfieldpopulation of P.
xylostella with already detectable resistance. A higher dose rate (twice of field
rate) of application and use of refuges (equivalent to 25% of area planted) was
found ineffective, once resistance was already detected. A refuge is intended to
dilute the resistance; however when the refuge is already highly contaminated
with resistance alleles, it can not be very effective in dilution. The outcome of
rotations of Bt-products also did not result on the anticipated lines.A switchover
134 Chapter 5

to Bt-aizawai was not found to improve the efficacy any significantly in overall
reduction of infestations of P. xylostella caused by Bt-kurstaki. However,
continued use of Bt-kurstaki would have further increased resistance beyond the
existing level. A likely explanation for these results given was that in the early
-
studies Bt-aizawai had shown 3-fold cross-resistance with Bt-kurstnki resistant
P. xylostella. Both Bt subspecies contain same CrylA toxins, whereas Bt-
niznwai alsocontains Cry 1C. Thusminimal cross-resistance may have
prevented Bt-aizawai from reaching a higher level of efficacy.

5.3 Mattagemertt of Insect Resistance by Designing Recombitrant


Bt Strains

Generally, resistance to B. thuringiensis insecticidal toxins is the consequence of


a mutation(s) that alters an insect midgut receptor proteins(s) so that it no longer
binds to the Cry protein. However, if a toxin gene was engineered so that toxin
bound to other midgut cell surface proteins, then resistance might be less likely
to arise (Glick and Pasternak, 1998). For example, the Raven@contains Cry3A
and Cry3Bb protein genes, in addition to two crylAc genes. The two cry3 genes
contribute to a much higher productivity in fermentation of this strain, and have
different binding characteristics on Colorado
potatobeetle
midgut cell
membranes. In a study conducted jointly by Ecogen scientists and Michigan
State University, laboratory-selected potato beetles, that were resistant to one of
the Cry3 proteins, showed only minimal resistance to the second Cry3. Thus, in
practice, an individual beetle would have to undergo two independent resistance
mutations to become resistant to the Raven strain. It was also seen that when the
same resistant potato beetle was exposed to a mixture of that Cry3 protein and
the CrylAc proteins, the Cry 3 resistance is strongly reduced (Figure 5.8). This
effect is presumed to be due to Cry-Cry interactions that occurs between the two

Cry3(1) resistant showed Minimal resistance to


Colorado potato beetle second type of Cry3 protein

1
exposed to

A mixture of
Cry3(1) and
strong reductim
in * Cry3 (1) resistance
Cry 1Ac

Figure 5.8 Two differentstrategiesforminimisingresistancedevelopment to


Raven Bt strain by Colorado potato beetle.
Insect Resistance to Bt Toxins 135

Cry proteins at the level of midgut binding. Thus, the Raven strain is claimed to
incorporate two different strategies to minimise the likelihood that the principal
insect target would develop resistance itto(Carlton, 1995).
Another approach of resistance management of Bt by recombinant
technology is by construction of hybrid protein construct. For example, the
insecticidal proteins CrylC and CrylE are both toxic to Lepidoptera but have
differentspeciesspecificities. CrylC isactiveagainst S. exigua,Mamestra
brassicae and Manduca sexta, while CrylE is active againstonly M. sexta. In an
experiment, hybrid CrylC-CrylE proteins were constructed and tested for their
toxicity to different insect species as well as for their ability to bind to different
receptors (Figure 5.9). The hybrid toxin G27, which contained domain I11 from
CrylC, wastoxicto S. exigua larvae,eventhoughitboundtothe CrylE
receptor but not to the CrylC receptor. Conversely, the hybrid toxin F26 was not
toxic to S. exigua larvae even though it bound to the CrylC receptor. Since the
CrylC andhybrid G27proteinsbindtodifferentinsectmidgutreceptors
althoughbotharetoxicto S. exigua, eithersimultaneousoralternating
treatments of S. exigua with these two Bt insecticidal toxins might limit the
development of strains that are resistant to these toxins. Resistance to both
CrylC andG27 would requiremutations in two separatemidgutreceptor
proteins (Bosch etal., 1994).

Domain
Domain
Domain Toxic to Competes for Competes for
I II 111 S.exigua CrylEsite
CrylCsite

CrylC + + I

CrylE 1 - - I +
G27 I I + I +
F26 I + I

Figure 5.9 Toxicity and binding specificity of CrylC, Cry1E, and hybrid
toxins G27 and F26 (Adapted from Bosch et al., 1994).
136 Chapter 5

5.4 Management of Insect Resistatice in Transgenic Bt-plants

Transgenic crops threaten the viability of Bt because, unlike the Bt sprays, which
last, but a short time, the crops produce toxins throughout their lives. This may
result in strongselection pressure with greater probabilityto resistance
development. McGaughey, Gould and Gelernter (1998) summarised the
strategies on insect resistance management to Bt-transgenic plants. The
strategies involved (a) decreasing the proportion of the pest population exposed
to a Bt- toxin by maintaining areas that are planted to crop cultivars that do not
produce any toxin. These areas serve as refuges for insect susceptible to the Bt-
toxins. These strategies also rely on (b) making sure that pests exposed to the Bt-
toxin encounter at least 25 times as much toxin as would be needed to kill 99%
of susceptible pest species, i.e. a dose expected to kill all or most heterozygotes.
In addition, (c) Bt-producing plants having two biochemically unrelated toxins,
each at ahighdose, is also expected to significantly decrease the rate of
resistance development (Figure 5.10). Many new strains of Bt, or Bt-toxins fit
the requirements for a multiple toxin strategy. However, many of these toxins
are similar enough in activity that resistance to one could cause adegree of
cross-resistance to others.
Liu and Tabashink (1 997) reported experimental evidence that a refugia
strategy, asasource of susceptible insects for mating withtheselected
populations, to prevent the fixation of resistance, delays insect adoption to Bt
toxins. Refuges to occur naturally, a certain percentage ofan agricultural
acreage is planted with non-transgenic plants. Rehges may also be established
by design through the use of seed mixtures or susceptible border rows. Menn
(1996) proposed mixing of transgenic with non-transgenic seeds(80:20) to
reduce the selection pressure for resistance in the target pestpopulations.
However, mixed seed strategy is controversial. A possible problem with mixed
seed arises from larvae that survive on a non-Bt plant and migrate to Bt cotton
where they will be less sensitive to Bt because of size. This could compromise
insect control and increase selection pressure for resistance by allowing the

7Managementofrefuges
Resistance
Management
Strategies
"t Application of ultra-high dose
( >25 times LCesof Bt toxin )

Presence of biochemically
unrelated mixtureof toxins
in flt producing plants

Figure 5.10 Resistance managementstrategies to Bf transgenic plants.


Insect Resistance to Bt Toxins 137

survival of heterozygotes. The likelihood of these occurring needs to be


determined experimentally before this strategy can be implemented. Monsanto
requires growers commitment to set aside a certain portion for refugla - either
4% non-insecticide treatedhon-Bt plants, or 20% insecticide treated, but not
with Bt - and they must agree to one of these strategies (Sims, 1996).

5.4. I Bt-Cotton

Introduction ofBollgard@cotton confers crop-resistance to some cotton pestsvia


the expression of Bt endotoxin and has provided excellent control of tobacco
budworm(Heliothis virescens). Nevertheless, the Bt-endotoxin is intrinsically
less potent against the related cotton bollworm (Heliothis zea) and this appears
to have led to some instances of bollworm damage in cotton not otherwise
treated. An over-spraying of Bt-cotton with the synthetic pyrethroid insecticide
A-cyhalothrin is reported to provide significant yield increases(Minketal.,
1997)). It was demonstrated ina laboratory study that insects that survived
exposure to sprayable Bt products at generally sub-lethal doses were susceptible
to h-cyhalothrin than unexposed pests (Harris, 1998). This study concluded that
oversprays of h-cyhalothrin on Bt-cotton tended to reduce the level of surviving
lepidopteran pests to very low levels. This vastly enhanced the dilution effect
offered by the influx of susceptible larvae from refugia andthus worked in
concert to offer a sustainable resistance management strategy(Broadhurst,
1998).

5.4.2 Bt-Corn

Resistance management in Bt corn is currently based on two complementary


principles: high dose and refuge. Bt corn is designed to produce very high levels
of Bt proteins, much higher than levels found in Bt insecticides (Ostlie et al.,
1997). The intent is to kill all European corn borer (ECB) larvae with no genes
for resistance, plus those with one copy of a resistance gene. In any population
of ECB, a few borers will have two copies of genes conferring resistance (rr),
some will have one copy of the gene (rs) and most will have none (ss). The
assumption inherent in this resistance management approach is that Bt hybrids
have achieved the high-dose objective. If the high-dose objective is not
achieved, then corn borer larvae with one copy of a resistance gene may survive
to adulthood and mate with other resistant moths. Most of the offspring from
these matings would be resistant to Bt corn. The second principle of the
resistance management plan is the use of refuges. The purpose is to provide a
source of ECBs, not exposed to Bt corn or Bt insecticides, to mate with potential
resistant moths emerging from nearby Bt corn. Thegoal is toproduce an
138 Chapter 5

overwhelming number of susceptible moths to every resistant moth. A refuge is


any non-Bt host, including non-Bt corn, potatoes, sweet corn, cotton or native
weeds that occur near Bt corn. Based on current knowledge of European corn
borer biology, pesticide resistance studies and computer simulation models, the
amount of refuge required has been estimated. It is approximately 20-30% of the
corn acreage, so that 20-30% larval population is prevented from Bt protein
exposure. This assumes that corn borers from alternative hosts emerge at similar
times as corn borers from Bt corn.

5.4.3 Bt-Potato

A resistance management program for transgenic potato, NewLeaf@, prepared


by the developing companyMonsanto has been accepted by
the US.
Environmental Protection Agency (Stone and Feldman, 1995). This includes, a)
incorporation of NewLeaf potatoes into IPM programs as an integral and not
stand-alonecomponent, b) monitoring of Colorado potato
beetle
(CPB)
populationsforsusceptibilitytoCry3Aproteinafterestablishingabase-line
susceptibility,c)highdoseexpressionofCry3A in potatoes to control CPB
heterozygous for resistance alleles, leaving only homozygous resistant beetles
witha ability to surviveNewLeafplants,d) rehgia, ashostforsusceptible
insects throughnon-transgenicpotatoes and e)searchanddevelopment of
Cry3A proteins with a distinct mode of action, use of multiple gene and alternate
gene strategies with a potential for substantially delaying or halting resistance.
Initially, Monsanto voluntarily placed a requirement on growers that no more
than 80% of their crop could be planted with the transgenic potato. In 1999,
EPA made it mandatory for plantationof20%structuredrefuge of non-Bt
potatoes in closeproximity to the Bt potatofield,aspartoftheregistration
(EPA, 1999).

5.5 Bt Resistance Genes and Estimation of Resistance Development

Tabashnik et
al.
(1997)
studied diamondbackmoth (Plutella
xylostella)
resistance to four Br toxins, i.e CrylAa, CrylAb, CrylAc, and CrylF. They
found that a single autosomalrecessive gene conferred extremely high resistance
to all the four Bt-endotoxins. This finding was significant, as Cryl toxins are
reported(Chambersetal.,1991) to have diverseinsecticidalactivityspectra
(Table 5.3), even though they have greater than 70% sequence homology. This
precludesthepossibilityofemployinganyof these single Cryl proteins
applications by rotation for delaying resistance development. As Bollgard cotton
contains CrylAc and transgenicBt corncontains CrylAb respectively,some
concerns have been raised that multiple resistance genes found in diamondback
Resistance
Insect to Bt Toxins 139

Table 5.3 Insecticidal activity spectra of differentCry1 protein toxins(Adapted


from Carlton and Gawron-Burke, 1993)

LC5, (rrg Cryl/nrn12diet sur$ace)


Cryl protei11
H V' H 2 OM SE"

Cryl Ab 0.68 16.9 0.27 38.8


Cryl Ac 0.04 1.4 0.1 1 >57

Cryl F 0.3 1 >5 7 0.27 25.6


(a) Heleliothrs virescetls (tobacco budworm), (b) Helicoverpa zea (corn earworm), (c)
Ostrirrra rrubilalis(European corn borer) and (d) Spodoptera exigua (beet armyworm).

moth may not be specific to these species only and that these genes may be
present in other lepidopteran species as well. The implication could be that a
polyphagous(insectsthat feed onmanycrops)lepidopteranlarvaesuchas
cottonbollworm (Helicoverpa zea), that developsresistance to CrylAc by
feeding on Bollgard cotton could become quickly resistant to CrylAb used in
transgeniccornor vice versa.Theseauthorsalsoreported that 21%of
individuals
fromsusceptible
a strain were heterozygous for
multi-toxin
resistance gene that meant that resistance allele frequency was 10 times higher
than previously established. This suggestedthat resistance might evolve to same
groups of toxins much faster than previously expected. However, Roush and
Shelton (1997) observed that the rate of evolution of resistance is much more
sensitive to changes in selection than in the initial resistance allele frequency.
Therefore it is difficult to arrive at a reliable initial resistance allele frequency in
field population from therates of resistance development in the field.
Gould et al.(1997)provided the first directestimateoftheinitial
frequency of Bt resistancegenesinsusceptible field populations of amajor
cotton pest, the tobacco budworm, Heliothis virescens. Resistant individuals are
rare initially, and it is inherently difficult to estimatetheirfrequencybefore
populations are exposed to an insecticide. The frequency results from a balance
between creation of resistant genotypes by mutation and selection against such
mutants when the insecticide is not present. Thus, 2000 male insects from four
cotton-growing areas were individually crossed with females of a strain selected
for its extreme high resistance to CrylAc, the Bt gene used in cotton against
tobacco budworm. Three males from the sample of 1025 successful crosses were
found to becarryingan allele for resistance to Bt toxin,that led Gould and
coworkers to estimate the field frequency of Bt resistance alleles to about 3 in
2000. Thisestimate is considerably higherthan thoseassumed in earlier
theoretical models and thus forebodes that resistance will evolve rapidly if pest
populationsreceiveprolonged and uniformexposure to Bt toxins.Thisalso
140 Chapter 5

implied that the success of the first generation of Bt-transgenic plants could be
short lived. Nevertheless,Gould et al. noted that if thecurrentresistance
management mandates (4% refuge) are followed, it should take at least 10 years
before resistance becomes a problem in Heliothis virescens, because of the
mortality of heterozygotes. This would not only outlast most intensive use of Bt
sprays on the diamondback moth, but also the historical average of chemical
sprays against cotton bollworms.

6 Concluding Remarks
Manyinsectpopulationshavebeenshowntoevolveresistanceto Bt 6
endotoxins in the laboratory, althoughonly P. xylostella hasdevelopedhigh
level resistance in the field. The mode of resistance to Bt in P. xylostella is
characterised bymore than 500-fold resistance to at least one CrylA toxin,
recessive inheritance, little or no cross-resistance to CrylC and reduced binding
of at least one CrylA toxin (Tabashnik et al., 1998). Analysis of resistance to Bt
in the diamondback moth and two other species (tobacco budworm and Indian
meal moth) suggested that this mode of resistance was common, althoughit may
not be the only means bywhich insects attained resistance to Bt.
Bt-based insecticides are frequently used in intensive agriculture, either
in conjunction with conventional insecticides asan Integrated Pest Management
strategy or as an alternative to synthetic insecticides against which resistance has
occurred. It is anticipated that the future will see a better balance to transgenic,
biologicalandchemicalmeansof insect control that reducestheselection
pressure against any one product. In this regard, the judicious use of chemicals
in combinationwith Bt-crops is likely toextendtheeffectiveness of this
technology in transgenic plants. isIt also anticipated that improved
understanding of the complex interplay among Bt toxins, their bacterial hosts,
their target organisms and the ecosystems they share will allow for the long-
term, effective use of Bt toxins for pest management.

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1993.Selection of aColoradopotatobeetle(Coleoptetra:Chrysomelidae)
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5 1. Wirth,M.C., G. P.Georghiouand B. A.Federici,1997.CytAenablesCryIV
endotoxins of Bacillus thuringiensis toovercomehighlevelsof Cry IV
resistance in themosquito, Culex quiquefasciatus.Proc.Natl.Acad. Sci.,
USA, 94, IO536 - 10540.
Part II

Viral Insecticides, Biofungicides,


Bioherbicides, and Mycoinsecticides
This Page Intentionally Left Blank
Natural and Recombinant
Viral Insecticides

1 Introduction
Demand for effective, more selective, safer pesticides, consistent with
sustainable agriculture continues to push the search for newer systems as well as
modifying the existing ones. Insects are subject to diseases caused by viruses,
bacteria, fungi, protozoans and nematodes. Many membersof these groups have
beenevaluated as biocontrol agents. Mostpathogensandnematodestend to
infect primarily insect larvae and are only effective against this stage. Although
bacterial control agents ofBacillus thuringiensis family are currently the leading
bioinsecticides, viral andfungalagentsaregraduallymakingheadway.For
example, baculoviruses directly compete with Bt for the control of lepidopterous
insects, albeit feebly at present. With the advances in development of
recombinant baculoviruses, that are more virulent and fast acting, baculoviruses
have a great potential for effective integration into pest management system.

2 Baculoviruses
Thebaculoviruses are the naturally occurring invertebrate-specific pathogens
that infect some important lepidopteran (butterflies and moths) hymenopteran
147
148 Chapter 6

(forest pests) insects. Their high pathogenicity, narrow host range and complete
safety to vertebrates and plants, makethem ideal candidatesforbiological
control of these insects. The viral pesticides can be applied using conventional
techniquesand do not create the problem associatedwith
residues.
Baculoviruses infect predominantly holomentabolons insects and almost all pest
species within the Lepidoptera are susceptible to infection by at least one of the
baculoviruses. Despite their potential, viral insecticides are employed much less
than they could be in crops and forests, due to difficulty in virus stability and,
mostimportantly,slowerspeed of actionthan that achievedwithchemical
pesticides.
Amongstthebestexamplesinvolving use of baculovirusesisthe
exceptional controlof velvetbean caterpillar(Anticarsia genzmatalis) on soybean
in Brazil and coconutrhinocerosbeetle (Otyctesrhinoceros) in Andaman
Islands in India.The Anticarsia gemnzatalis nuclear polyhedro virus (AgNPV)is
highly virulentto hostlarvae at alowdose(1.5 x 10"OB/ha)and it is
efficientlytransmitted in the hostpopulation by naturalenemiesandabiotic
factors. As a result, one AgNPV application per cropping season is sufficient to
control the insect pest and is as effective as two insecticide applications in a
season. As aleaf feeder, A.gelnlnatalis is exposed to appliedAgNPVand
usually no other key insect occurs simultaneously on the crop. Also, soybean
tolerates considerable defoliation with no reduction in yield (Moscardi, 1999).
Another successful application of baculovirus has been the control of coconut
rhinocerosbeetle (0. rhinoceros), serious
a pest that threatens
coconut
production. The application of baculovirus effectively controls this pest at a low
cost. The strategy employed in the Andaman Islands in India, has been to induce
virus epidemics in beetle populations, in the newly invaded areas, by infecting
andliberatingbeetleswith Oryctes baculovirus.Resultscomparinglevels of
beetle damage to coconut, before and after virus introductions, have been 80-
90%reductions(Jacob,1996).Onceintroduced, the virusseemstomaintain
itself adequatelywithout additional intervention.
A number of recent reviews on naturally occurring baculoviruses have
appeared in the literature (Federici,1999;Moscardi,1999;Volkman,1997;
Hammeretal.,1995;Cunningham,1995; and CrookandJarret,1991). The
developmental efforts on recombinant baculoviruses have also been reviewed
(Treacy,1999;Possee,1997;Bonning and Hammock,1992,1996;Hooveret
al.,1996;Maeda,1995;andWood,1995). An overviewoftheprogress
achieved in realizing the potential of natural and recombinant baculoviruses has
been provided in this chapter.
nsecticides
Recombinant
Viral
and
Natural 149

2.1 Classification

The baculoviruses are divided into two sub-groups based on their morphology.
In sub-groupA, Nuclear Polyhedrosis Viruses (NPVs) have many virus particles
embedded in each occlusion body, which are called polyhedra becauseof their
shape. Some NFVs have a single nucleocapsid within each virus particle and are
designated SNPVs. In contrast the h4NPVs have multiple nucleo-capsids within
each virus particle. These have been isolated from several hundred insect species
including Spodoptera littoralis, S.jkgiperda, Lymantria dispar, Helicoverpa
zea and Heliothis armigera. The NPVs are named after the insect species from
which they were first isolated, followed by the appropriate baculovirus sub-
group name.
In sub-group B, Granulosis viruses (GVs) have a single virus particle
embeded in each occlusion body, which is capsule-shaped and called granules.
Granulosis virus (GV), does not grow well in cell culture, thus interest in their
industrial exploitation has been negligible. However, GVs can be used against a
number of pestspecies,including Pierisbrassicae oncabbagesand Plodia
interpunctella, a stored product pest.

2.2 Characteristics

The baculoviruses are characterized by the presence of rodshaped nucleocapsid


6.1) to form the
which are further surrounded by a lipoprotein envelope (Figure

Figure 6.1 Electron micrographs of (a) Baculovirus particles or polyhedra


(b) Cross-section of polyhedra and (c) Diagram of polyhedra
cross-section (Courtesy Jean Adams andV.DAmico)
150 Chapter 6

virus particles. Certain baculoviruses, specifically nuclear polyhedrosis viruses


(NPVs)haveaunique life cycle that involves the temporallyregulated
expression of two functionally and morphologically different viral forms, the
budded form and the occluded form. Nuclear polyhedrosis viruses producelarge
polyhedral occlusion bodies, which contain enveloped virus particles within the
nucleusofaninfected cell. Theocclusionbody is composedofamatrix
comprisinga29kDaprotein known aspolyhedron.Theseocclusionbodies
serve to protect the virus particles in the environment and also provide a means
of delivering the virus particles to the primary site of infection in insects, the
midgut epithelial cells. Thebuddedformof the virus is involved inthe
secondary infection, virtually affecting all the tissues within the host larvae.
Replication of the virus in other organs creates extensive tissue damage and
eventually death. When an infested insect dies, millionsofpolyhedraare
released. This allows the virus to persist in the environment although its host
insect larvae may be present for only a few weeks duringthe year.
The pathogenicity of baculoviruses has been characterized mostly by
their median lethal dose (LDso),an estimate ofinfectivity of the virus to its host.
Virulence, which in the case of baculovirusesis best determined by the speed by
which a given virus elicits the desired response, is an important measure of the
effectiveness of the viruses as a biopesticidal agent (Van Beek and Hughes,
1998).

2.3 Nuclearpolyhedrosis viruses (NPVs) - Potential as Viral Insecticides

Nuclear polyhedrosis viruses are being developed for control of lepidopterous


larvae. In the USAandEurope,afewbaculovirusproductsareproduced
commercially for use in field crops (Table 6.1). The companies like Dupont,
Biosys (now Thermo Trilogy), American Cyanamid and Agrivirion (to name a
few) have active research programs for development of agricultural-use viral
insecticides. For
example,Biosys
had
introduced
two
baculovirus-based
products, Spod-X for control of beet armyworm and Gemstar LC for control of
tobacco budworm and cotton bollworm.
The limited commercial penetration of the market by viral insecticides
suggests that major factors constrain their wider use. Mostimportantly,
baculoviruses compete inadequately with classical insecticides because of the
viruses' slowspeedof kill. Thetimeperiodbetween the initial infection by
baculoviruses to the death of diseased larvae varies fiom 213 days to 213 weeks,
depending on the virulence of virus isolate and nutrition of larval host. Further,
an NPV-infected larva continues to feed until time of death; thus continues to
damage the crop after treatment.Thereforemostcommerciallysuccessful
baculoviruseshavebeenusedprimarilyoncropswhichcan sustain damage
without major economic losses, such as forests (Possee et al., 1997). Some of
nsecticides
Recombinant
Viral
and
Natural 151

Table 6.1 Commercialbaculovirusformulationsforuse in fieldcrops

Product Name PestsKrops


Baculovirus
(Manufacturer) -

Spod-X Spodoptera exigua Beet armyworm of


(Thermo Trilogy) Nucleopolyhedro Virus floral crops
(SeNP V)
GemStar (ThermoTrilogy), Helicoverpa zea Several Heliothisl
Elcar (Novartis) Nucleopolyhedro Virus Helicoverpa spp.
(HzNPV) on cotton, corn,
tobacco, sorghum,
soybean and tomato
Cyd-X, (Thermo Trilogy), Cydia pomonella Codling moth in
Madex (Andermatt Biocontrol, Granulo Virus walnuts, apples, pear
Switzerland), Granusal (Behring (CPGV) and plum.
AG, Werke, Germany),
Caprovirusine (NPP, France)
Mameshin Matnestra brassicae Cabbage moth,
(National Plant Protection, Nucleopolyhedro Virus American bollworm,
France) (MbNPV) diamondback moth.
VPN Anticarsia gemnatalis Velvetbean caterpillar
(Agricola El Sol, Brazil) Nucleopolyhedro Virus on soybean
(AgNPV)
Gusano Autographa californica Alphalpha
looper
(Thermo Trilogy) Nucleopolyhedro Virus
(AcNPV)
Spodopterin Spodoptera littoralis Cotton
and
vegetables
(National Plant Protection, Nucleopolyhedro Virus
France) (SlNP V)

the baculovirus products for control of forest pests have been registered by The
U. S. DepartmentofAgricultureForestServiceandCanadianForestservice
(Table 6.2).
In most cases, it is difficult to develop cell cultures which will support
replication of viruses, particularly that of baculoviruses. However, there are two
widehostrangebaculoviruses that are easytoworkwithandhavegood
potential for beingproducedbyfermentation.Oneofthese is Autographa
californica multiple nuclear polyhedro virus (AcMNPV), for which very good
cell cultures havebeenavailableandtherefore it is well studied. This virus,
originally isolated from the alfalfa looper is of particular interest for insect pest
management. The virus is known to infect 39 species of Lepidoptera, including
152 Chapter 6

Table 6.2 Commercial baculovirus products for control of forest pests

Product Crop Virus Target

Gypchek Diaspora
Lymantria Broad-leaved
Gypsy moth
trees Virus Nucleopolyhedro
(LdNPV)
TM BioControl-1,
Orgyia
pseudotsugata
Tussock
moth
Douglas fir
Nuclcopolyhedro
Virtuss Virus
(OPNPV)
Virox Neodiprion sertqer Spruce saw fly Pine
Nucleopoyhedro Virus
(NeseNPV))

those of the major pests Heliothis, Spodoptera and Trichoplusia (Cunningham


1995).
Another multiple nucleopolyhedro baculovirus is that of the celery
looper, Anagrapha falcifera (Kirby) (AfMNPV), that has also shown potential
for development as a microbial insecticide. Much like AcMNPV, the AtMNPV
has a broad host range and infects 3 1 species of Lepidoptera in 10 families
(Hostetter and Putler, 1991). In a comparative field study, AfMNPV has shown
greater virulence than Heliothis NPV toward H. Zea on corn ears. It was also
found that AfMNPV application reduced damage and ear protection was
comparable or betterto Btk (Javelin@) and Bt-aizawai (XenTari@)products
(Pingel and Lewis, 1997).

2.4 Mode of Action of Baculoviruses

Viral infection debilitates the host larvae resulting in reduction of development,


feeding and mobility and increasing exposure to predation.On ingestion of
polyhedra by insect larva, the polyhedra move to the midgut where the alkaline
environmentandpossibly proteolytic action,dissolve the polyhedrincoat
releasing the infectious nucleocapsids. The released nucleocapsids bind to the
midgut epithelial cells, migrate through the cytoplasm and uncoat in the nucleus.
Viral replication in the nucleus produces progeny nucleocapsids that bud
through the plasma membrane of infested cells into the circulatory system of the
insect. The open circulatory system of the insect provides the virus with access
to other tissues within the host larvae. Ths results in secondary infection of
tissues throughout the insect. Nucleocapsids may then be enclosed within the
polyhedra in the nucleus, or continue to spread infection within the larval host,
as shown in Figure6.2.
Natural and Recombinant Viral Insecticides 153

The baculovirus is sprayed


onto the foliage

the baculovirus DNA


Baculovirus DNA dissolves and the DNA
is spread through cellsstomach
enters the

v i ~is ~replicated
B a ~ ~ l oDNA
by stomach cells until the
stomach cells rupture . The
caterpillar stops feeding

Figure 6.2 Mode of action of baculoviruses (Adapted fiom Georgis, 1996).

Post-larval effects may include lower pupal and adult weights, as well
as reduced reproductive capacity and longevity. The polyhedra that are1-10 pm
in size account for up 30% to of thedry weight of the infected larvae. There may
be up to 30 ormorepolyhedrain infected cellsandlateinstarlarvaemay
produce up to 10" polyhedra before death. The insect may continue to feed till
deathoccursfiom viralinfection,dependingonthevirusconcernedand
environmental factors. Infected defoliating larvae usually climb to the upper
parts of the plants, dying in 5-8 days, depending on biotic and abiotic factors.
Upon death, these are seen hanging limply from foliage and have a characteristic
shiny-oily appearance, Figure6.3.
Park et al. (1996) have studied the baculovirusLymannia dispar NPV
and found that it interferes with insect larval developmentby altering the host's
hormonalsystem.The level ofhemolymphecdysteroids,theinsectmolting
hormone, was higherin virus-infected larvae than in uninfected controls. There-
Chapter 6

D.

Figure 6.3 Virus killed caterpillar


(Courtesy Roger T. Zerillo, ref.33).

fore, it is likely that the insects are no longer under the control of the hormonal
system after infection by viruses. Volkman (1997) observed that there is a fierce
competition at a molecular level between the baculovirus and its host over the
final disposition of the resources of the hosti.e. whether they go toward building
polyhedraoramoth.Thehigherlevelofecdysteroidpreventssubsequent
conversion of larvae to moths, while at lower concentrations molting proceeds
normally.

2.5 Production of Baculoviruses

Viruses are facultative parasites and will only replicate in living cells, making it
necessary for these to be produced either in host insect larvae (in vivo) or in
susceptible cell culture (in vitro). Until recently, NPVs have been produced in
vivo and harvested from insect cadavers and processed as powders. This method
whilst adequate on a small scale, is costly to scale up and difficult to assess for
quality control in a commercial environment. Recent advances in tissue culture
technology and deep fermentation should enable cheaper production of NPVs.
Chakraborty et al. (1995) reported in vitro production of wild type Heliothis
baculoviruses.Theviruses,including Heliothiszeu and Heliothisarmigeru
NPVs in a H. zea cell line, adapted to grow in suspension culture using serum
free medium. Several companies are also pursuing this technology. In addition
Crop Genetics International(now Therm0 Trilogy))and AgriVirion (New York,
N Y ) have recently developed more cost effectivein vzvo production systems. It
is anticipated that these advances will significantly reduce the production costs
of viral pesticides.
Natural and Recombinant Viral Insecticides 155

2.6 Formulation of Baculoviruses

Themajority of thebaculovirusesareformulatedasconcentratedwettable
powders. The corn earworm (Helicoverpa zea) NPV biocontrol product Elcar
(Novartis) is formulated by diluting with an inert carrierand spray drying or air-
drying.Formulation of Anticarsiagemmatalis NPV with amorphoussilica,
attapulgite and kaolinite clays were found to maintainthe activity after a year of
storage, while in the presence of bentonite, activity declinedto 50% in the same
period(Medugno et al., 1997). H.zea NPV forcontrol of H.zea and H.
virescens in cotton is produced as a liquid concentrated formulation GemStar"
by Thermo Trilogy. On the other hand, gypsy moth(Lymanrria dispar L.) NPV
arefreeze-dried,either with carbohydrate
a or
by acetoneprecipitation,
Additives are frequently put into tank mixes, which can improve the physical
performance of the baculovirus spray deposit. A typical formulation consists of
adjuvants-surface active agents such as wettingand dispersing agents, spreaders,
stickers, sunlight screens, buffers and also gustatory stimulants (for information
on gustatory stimulants, see Chapter4).
The
abioticfactor that is most responsible for inactivation of
baculoviral occlusion bodies in natural environment, is sun light; particularly
wavelengthsof290-320 nm caninactivatemostviruses.Nucleopolyhedro
viruseshavebeenreportedtobeevenmorephotolabile than other insect
pathogens, such as Bt. Presence of free water, for example, dew deposits on
foliage,canacceleratedestructionofNPVs by sunlight.Recentadvances in
protecting NPVs from photo degradation through the adoption of sun screening
agents and opticalfluorescentbrightenershas increased their utility. Several
effectivedyes,suchaslissaminegreen,acridine yellow, alkaliblueand
mercurochrome have been used asphotoprotectants(Shapiro,1995).Optical
brighteners also absorb UV light and have been shown to significantly reduce
photodegradation of NPVs and enhance viral activity. The addition of stilbene
oxide brighteners has been shown to increase the efficacy of the NPV of the
gypsy moth (Lymantria dispar). These substances have enhanced the efficacy of
other baculoviruses, including the NPVs of H. zea, H. virescens, A. californica,
S. exigua, T. ni, C. includans,A.falciffra and A.gemmatalis. A similar
enhancinginfluence of afluorescentbrightenerCalcofluor white M2Ron
nuclear polyhedrosis virusof Anagraphafalcifera (AMNPV) has been reported
with reduced LTso values on four noctuld pests (Vail et al., 1996). The possible
mode of action for these compounds when combined with vinlses in the insect
gut,hasbeendescribed(Washburn et al., 1998).Thebrighteners may block
sloughing of infected primary target cells in the insectmidgut and thus affect the
host susceptibility to the baculoviruses.
156 Chapter 6

2.7 Enhancement of Viral Pesticidal Activity by Enhancin

The discovery of NPV enhancing proteins as synergists for NPVs adds another
dimension to the utility of NPVs as insect control agents. A 104 kDa protein
called Enhancin (formerly known as viral enhancement factor, VEF) contained
in the granules of occluded i? ni GV, increases the viral pesticidal activity in
noctuid moths at levels of 50 pic0 gram per insect. This protein appears to
comprise about 5% of TnGV polyhedra (Gallo et al., 1991). The biochemical
activity,heldwithinthepolyhedronproteinmatrixandreleasedduringthe
alkalinedissolution of polyhedra in
the
insect
midgut causes
specific
biochemical and structural changes in peritrophic membrane (the interior lining
of the insects intestine). The peritrophic membrane, which is made mostly of
chitin and proteins, is an important component of the insects immune system
and serves as a protective barrier that separates the insect midgut from the
infectionsusceptiblemidgutepithelicalcells.Enhancinbindstoamajor
mucinous protein of peritrophic matrix, destroying the matrix structure, thus
allowingthebaculovirustopassthroughtherelativelyimpermeablematrix
unimpeded, resulting in the rapid death of the insect (Wang and Granados,1997)
(Figure 6.4).

Intestinal
peritrophic
Mucinous cells

A
testinal cells

lallClll \/W ,
Mucinous peritrophic
matrix barrier
B

Figure 6.4 (A) Virusescannotpass throughIntestinewallto kill insect.


(B) Enhancinproteinattackstheimmune system allowing
viruses to pass through (Courtesy: Granados, 1997).
Recombinant
secticides
Viral
andNatural 157

In dose-responseassays with T. ni larve, inoculatedwithAcMNPV,


TnSNPV andAwticnrsin gemtlatalis MNPV, the addition of enhancinresulted
in 3- to 16-fold reduction in LDSos.Thus, the addition of supplemental enhancin
in baculovirus pestmde products could significantly reduce applicationrates and
would be partlcularly useful for the control of late Instar larvae, that typically
require very high application rates to catch infection.
Menn (1996) has also quoted unpublished research works suggesting
that NPVs could be synergistically combmed with reduced rate of conventional
insecticides for insect control (for detailed discussion see Chapter 10).

3 Genetically Modified
Baculoviruses
for Insect Control
Biotechnology, through recombinant DNA technology has provided the means
of overcoming some of the shortcomings of naturally ocurr~ngbaculovlruses,
while maintaining or enhancing their desirablepest-specificcharacteristics.
Nucleopolyhedroviruseshavebeengeneticallyaltered to enhance the speed
with which they kill the target pest.This has beenachievedthroughgenetic
manipulationofbaculoviruses by exchange of genetic material between
different baculovimses or insertion of foreign genesinto baculovirus genomes.
Baculoviruses are rod-shaped particles containmg at least 25 different
polypeptides and a large (100-200 kbp) circular double stranded DNA genome.
The NPVdouble-stranded DNAis highly amenableforDNArecombinant
technology through the use of the polyhedrin promoter gene that 1s suited for
expression of exogenousgenessuchas neurotoxins, neuropeptides,juvenile
hormone esterase andothers.
Two major baculovms expression systems have been developedfor the
production of recombinant proteins. These are based on nucleopolyhedro viruses
derived from alfalfa looper (Aurogrnphn c(z1ijornictz) and a similar virus from
the silkworm ( B o r ~ h ymori).
,~ The entire sequences for the genomeofboth
Autogrnplln cal<fornicaNPV (AcNPV) and Bombzvx tnori NPV (BnlNPV) have
beendetermined. The genome of AcNPV has a double-strandedcovalently
closed circular DNA of 134 kilobase (kb) pairs.
Recombinantbaculovirusesareconstructed in twostagesdueto the
difficulty of manipulating the large genomedirectly. The foreigngene is
lncorporated initdly into a baculovirustransfervector. The gene-inserted
plasmid is thenpropagated in the bacterium Escherichicr coli. Most transfer
vectors used are bacterial plasmid University of California (pUC) derivatives,
whichencodeanorlglnof replication forpropagation in E.coli and an
ampicillin-resistance gene. The pUC fragment 1s ligated to a small segment of
DNA taken from the viral genome. The foreign gene sequence is lncorporated
into a cloning site downstreamof the promoter selected to driveexpress~on.For
158 Chapter 6

3c- <
3
- Gene of interest

-
0 Baculovirus
partides

Baculovirus DNA
(-1 50 kb)

insect cells containing


Plasmid
gene of interest

Culture cells
l
i
Recombinant
virus M\IA

Plasmid recombines Recombinant


into viral DNA baculovirus

Figure 6.5 Construction of recombinantbaculovirus

the second step, the transfer vector is mixed with DNA from the wild-type
baculovirus. The engineered DNA is incorporated into the virus via homologous
recombination events within the nucleus of cultured insect cells (Figure 6.5).
The baculovirus system allows the precise insertion of foreign DNA without
disruption of other genes, unlike genetic engineering in plants, which results in a
ratherrandomincorporationofnewDNAintothegenome(Bonningand
Hammock, 1996).
Any gene coding for a protein that disrupts normal larval development
or behavior and reduces feeding damage caused by the insect is a candidate for
expression by a recombinant baculovirus for insect control program. Production
duringthe
infection
ofproteins
that
interferesspecifically
with
insect
metabolism or metamorphosis such as hormones, enzymes and toxins, might
Recombinant
and
Natural Viral Insecticides 159

enhance the pathogenicity of these viruses. Theproduction of a diuretic


hormone, a mite toxin, a scorpion toxinor a modified juvenile hormone esterase
enhances biological activity of baculoviruses and so these are the first examples
of genetically improved baculovirus insecticides (Table 6.3).
The recombinant virus expresses the genes for rapid kill of the host and
is subjected to strong negative selection pressure. Aquickerdeath almost
certainly would lead to fewer viral progeny. Resultantly, recombinant virus can
not compete with its wild type counterpart in the field. Recombinant
baculoviruses, therefore are being developedasbiological insecticides for
repeated application rather than as biological control agents that establish and
recycle in the field.

3.1 RecombinantAcNPVExpressingToxins

Arthropod venoms offer a rich source of insect-selective toxins. In fact,


expression of insect selective toxins in the baculovirus expression system has
proved to be highly successful for increasing virus efficacy in insect pest
control. Baculoviruses expressing the insect specific toxin AaIT derived from
the Algerian scorpion(Androctonus australis) areamong the mostpromising
recombinant baculoviruses developed yet. This neurotoxin which does not have
any effect on mice, disrupts the flow of sodium ions in neurons of targeted
insects, causing repetitive firing of motor nerves and overstimulation of skeletal
muscle. Larvae infected with these recombinant baculoviruses exhibit dorsal
arching and increased irritability and cessation of feeding. Lethal timesare
reduced by 25-40% compared with those of the wild-type virus. The feeding

Table 6.3 Genes that increase the insecticidal effects of baculoviruses


(Adapted from Maeda, 1995)

Ef/ect(s) of expressed gene o/o increase i n


Nanle of Gene
011host insect irlsect killing

rDisrupts
hormone
Diuretic 20%
Feeding cessation (1 '' instar) and
Juvenile hormone esterase
blackening
FeedingBt toxin
ation 2 X LDSo
Paralysis, body tremors and dorsal
Scorpion toxin (AalT) 30-40%
arching
Mite (Txp-I) Paralysls 30-40%
Wasp toxin Premature melanization and low
(Antigen 5 ) weight gain
160 Chapter 6

damage by T. ni larvae infected with recombinant virus is reduced by 50% on


cabbagecomparedwithdamagecausedby larvae infectedwithwild-type
viruses. When AcNPV.AaIT was tested under field conditions, it was found to
be even moreeffective. In the field, it killed the insect pests faster, decreased the
damagetocabbage plants andreduced the secondarycycleof infection
compared to the wild-type virus (Cory et al., 1994).
It is reported that speed of virus kill further increases in the presence of
low doses of pyrethroid insecticides. Pyrethroid resistant insects are also killed
more quickly by this combination. Since AaIT has a similar mode of action to
the pyrethroid insecticides, the possibility of cross-resistance to the toxin was
investigated. Instead of being susceptible to cross-resistance, AcNPV.AaIT was
found more effective against resistant larvae than pyrethroid-susceptiblelarvae.
Two other neurotoxin genes that have been widely incorporated into
the AcNPV genome are LqhIT2, which encodes a venome component of the
yellow scorpian (Leirus quinquestriatus hebraeus) and Tox34,which expresses
TxP-I toxin derived from the straw itch mite (Pyemotes tritici). Unlike AaIT,
which is excitatory, LqhIT2 isa depressant-typetoxin resulting inflaccid
paralysis of the virus-infected insect. Thestraw itch miteusesatoxinto
immobilize insects up to 1,50,000 times in size by causing muscle contraction
and paralysis. TheAcNPVscarrying either LqhIT2orTxP-Ihavebeen
evaluated against lepidopteran larvae, with each found to induce significantly
enhanced speed of mortality (lethal time 30-40% less) than wild-type AcNPV.
Thus, this engineered baculovirus not only hastened the demise of the infected
insect larvae, it also significantly decreased the ability of insects todamage
plants. These successes
with
toxins of relatively limited activity on
lepidopterous larvae suggest that insect-selective peptides of far greater activity
will prove useful with currently used expression systems.
Due to limited, effective hostrangeofrecombinantAcNPV, its
potential to provide acceptable pest control might have utility against only two
most AcNPV-permissive pest species, T. ni and H. virescens. One way to design
a recombinant baculovirus with a more desirable target profile is to insert a toxin
gene into a wild-type virus, which naturally exhibits high levels of pathogenicity
to the pest complex one needs to control. For example HzNPV recombinants
were recently engineered to carry either the LqhIT2 or AaIT toxin gene. These
recombinants are potentially more effective biopesticide than AcNPV. AaIT for
control of heliothine complex in cotton (DuPont, 1997; American Cyanamid,
1997)

3.2 Recombinant AcNPVExpressingInsectHormones

Expressionofacomponentofthe insect endocrinesystem in recombinant


baculoviruses is rationalized onapremise thatin the eventofa single
Recombinant
and
Natural Viral Insecticides 161

component is overproduced, homeostasis will be disrupted, causing a deleterious


effect on the insect. To test this possibility, recombinantAcNPVswere
producedexpressingeclosinghormone(which is involved in ecdysisand
sheddingof the cuticle duringmolting)and the prothoracicotropichormone
(PTTH) from B. mori (which stimulates production of ecdysone), respectively.
However, none of these recombinant baculoviruses showed much enhancement
in efficacy as compared to wild-typevirus.

3.3 Deletion of egt Gene from AcNPV Genome

Insecticidal activity of the baculovirus can also be increased by the deletion of a


gene(s)from the viral genome that alters the normal life cycleofthe insect
larva, but is not essential for virus replication.
ThebaculovirusAcNPVproducesanenzymeecdysteroidUDP-
glucosyl-transferase (est), which inhibits or delays molting and pupation in the
host larvae. Expression of this protein prevents the host insect from molting and
effectively keeps the insect in the feeding state. The deletion of the egt-gene
from the viral genome allows the infected larvae to begin molting, which results
in feeding cessation i.e. an increasein insecticidal activity, as compared to wild-
type AcNPV-infected insects, which continue to feed throughout the period of
infection. It was demonstrated that deletion of the egt-gene of AcNPV caused
infected fall armyworm (Spocioptera fiugipercia), a 10-20% reduction in lethal
time, a 40% reduction in feedingdamageanda 30% reduction in progeny
viruses produced (OReilly andMiller, 1991).
In a field trial, Treacy et al. observed that while egt-deleted AcNPV
provided more consistent control of T.ni on lettuce and cabbage, differences in
efficacy between the wild-type and egt-deleted AcNPV were marginal. They
concluded that any further changes that might be made in the baculovirus
genome (e.g., gene insertion), to enhance killing speed beyond that seen with
egt-deletion, shouldproduceamorecommerciallyacceptablebiopesticide
(Treacy, 1999).
AmericanCyanamid carried out field trials using cotton, tobacco,
lettuce and cabbage as crops. A cotton trial in 1996, showed that treatment with
recombinant virus egt-minusAcNPVcontaining the AaITaloneproduced
controlof Heliothis virescenswhichwasequivalent to that in achemically
treated control plot with acephate (American Cyanamid, 1997).
The concept of gene-deletion construct is more easily acceptable than
for exampleagene-addition construct, bytheregulatoryagencies.The egt-
deleted recombinant AcNPV was the first genetically modified baculovirus tobe
field tested in the United States. Thus, it is likely to be the first recombinant
baculovirus approved for commercial use, as a bio-pesticide.
162 Chapter 6

3.4 Recombinant AcNPVExpressing Juvenile Hormone Esterase

Insects undergohormonicallycontrolledmetamorphosisduringdevelopment.
This aspect of insect biology has been exploited for the development of faster-
acting recombinant baculoviruses.
Juvenile hormone (JH) is intrinsically involved in regulation of gene
expression in both larval and adult insects. From late embryonic life through the
final larval instar, juvenile hormone is produced and secreted and maintained at
measurable levels, resulting in the retention of larval characteristics. The
reduction in JH titer is a key eventin insect development that leads ultimately to
termination of feeding and metamorphosis from larval to pupal stage. Juvenile
hormone esterase (JHE) increases as JH titer decreases. The expression of JHE
in a baculovirus vector, is to function as an anti-JH agent.
Thecodinggene for JHEfrom the tobaccobudworm (Heliothis
virescens) was expressed in various baculovirus constructs, including AcNPV.
Despite high levels of expression of JHE, no significant improvement of the
insecticidal efficacy was observed for neonate larvae of T. ni. or other insect
species tested.

3.5 Environment Risk Assessment of Recombinant Baculoviruses

The potential hazards associated with chemical andviral pesticides differ mainly
in
the fact that viral pesticides replicate and, therefore, environmental
contaminationcanincrease in concentrationandarea.Aseriousconcern is
whether or not a recombinant protein, such as a toxin produced in the virus-
infected insect, will presentahazardtootherspecies in the environmentby
persistence. This carries an additional concern because once established in the
environment, there is a finite probability that it cannot be removed if that is
desired. Inorder to assess the risk of the release ofageneticallymodified
organism,geneticmodificationsneed to be evaluated in relation to two key
ecological and environmentalissues:
(a) In what respect do the modified and wild-type baculoviruses differ in
their effects on susceptible species and populations? and
(b) Has the genetic modification altered the capacity of the baculovirus to
persist and spread in the environment

Considerable research has been conducted to determine both absolute


and relative impactofAcNPV-AaITwithrespecttowild-typeAcNPV, on
survival of non-lepidopteran invertebrates. All the studies reported indicate that
atleast this particular recombinant baculovirusexhibits the same host rangeas its
wild-type counterpart. The range of virulence exhibited by AcNPV-AaIT, even
in Lepidoptera, appears to be unalteredas compared to wild-type AcNPV. It has
Recombinant
secticides
Viral
andNatural 163

been shown that dosage-mortality responses were similar for AcNPV-AaIT and
wild type AcNPV (Treacy, 1999).
Fuxa et al. (1998) tested the capability of recombinant baculoviruses to
compete with a wild-type baculovirusfor a host-insect resourcein a green house
microcosm.Tworecombinantbaculoviruses,AcNPVexpressingascorpian
toxin(AcNPV.AaIT)andAcNPVexpressing the mutantformofan insect
derived juvenile hormone esterase (AcNPV.JHEKK) were tested with a wild-
type Autographacalifornica nucleopolyhedrovirus(AcNPV.WT) on cabbage
looper (Trichoplusia ni) larvae. They found that AcNPV.WT out-competed the
recombinantviruses for aniche in agreenhousemicrocosm.Thisfinding
supports that the foreign genes suchas AaIT is of negative value tothe virus and
probablyensuresacompetitivedisadvantageofrecombinantbaculoviruses
persisting in the agro-eco system relative to wild-type AcNPV.

4 Concluding Remarks
Recombinantbaculovirusesrepresentavaluabletechnology that has great
potential for effective integration into pestmanagementsystem.Both the
recombinantandwild-typevirusesaremoreenvironmentally attractive than
chemical pesticides and may represent a major step forward towards a more
sustainable agriculture. Much of the research directed at improving pesticidal
propertiesofbaculoviruseshasbeenconductedwithAcNPV.However,
recombinant AcNPV may have limited effective host range and utility against
only pest species such as T. ni and H. virescens. This would present a barrier to
its commercialization.Arecombinantbaculoviruswithmoredesirable target
profile would be the one, which exhibits high levels of pathogenicity to the pest
complexsuch as for the controlof the heliothinecomplex in cotton. For
example, HzNPV recombinants carryingeither LqhIT2 or AaIT toxin-gene have
potential to be more effective biopesticides for pest control in cotton.
Commercialization potential of recombinant baculoviruses technology
at the current level couldbe attained by its deployment in rotational-use
strategies in controloflepidopteranpest species, for relaxing the selection
pressureexertedby synthetic pesticides oreven Bt on the pestpopulations.
Thesecould also be utilized in binary mixtures
with
certain
chemical
insecticides. Augmentationoftransgeniccrop varieties andestablishmentof
binary transgenic host-plant resistancehiocontrol system could provide another
use for them.
Optimizationof viral shelf life, better suspensionof the virus and
increased stability under field conditions are some of the other challenges that
would require satisfactory resolution by formulationtechnologists.
164 Chapter 6

References

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genetically engineered microbial pesticide, in Federal Register, United States
Environment Protection Agency, Washington, DC,62, 39,5 18.
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genetically
engineeredviral
insecticides, Biofechnology and Getlefic
Engineering Reviews, IO, 455-489.
3. Bonning, B. C.and B. D. Hammock,1996.Developmentofrecombinant
baculoviruses for Insect Control, Annu. Revs. Entonlol. 41, 19 1-210.
4. Chakraborty, S., A. Kanhalsingh, P. I. Greenfield, S. Reid, C. Monsour and R.
Teakle, 1995. I n vifro production of wild-type Heliothis baculoviruses for use
as biopestlcides, AustralasianBiotecAnoI., 5(2), 82-86.
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M. Carty. R. D. Possee, P. J. Cayley and D. H. L. Bishop, 1994. Field trial of a
genetically improved baculovirus insecticide, Nature, 370, 138-140.
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Approaches to Integrated Pest Management, R. Reuveni, ed., CRC Press, Boca
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testing of geneticallyengineeredmicrobialpesticides, in Federal Register,
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23,449.
9. Federici, B., (1999). Naturally
occurring
baculoviruses
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insect
pest
control, in Biopesticldes: Use and Delivery, F. R. Hall and J. J. Menn, eds.,
Humana Press, Totowa, NJ, pp. 301-320.
10. Friedlander, B. P., Jr., 1997.Newbiologicalcontrolofinsectpests is
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insect, Cornell University Science News, http://www.news.corneIl.edu/Science
lJune97lEnhancin Granados.bpf.htm1
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Capability
recombinant
of insect
viruses
environmental
for
persistence/transport, http://nbiap.biochem.vt.edu/brarg/brasym9b/fuxa96 htm.
12. Gallo, L. G., B. G. Cossaro, P. R. Hughes and R. R. Granados, 1991. In vivo
enhancement of baculovirusinfection by theviralenhancingfactorofa
granulosis
virusof
the
cabbage
looper, Trichoplusia n i (Lepidoptera:
Noctuidae), J. Invertibr. Pafhol.,58, 203-210.
13. Georgis, R., 1996.Presentandfutureprospects of biologicalInsecticides,
Cortference in Biological Control, Cornellcommunity,April, 1 1-1 3,1996,
http:l/www.nysaes.comell.edu/ent/ bcconf/talks /georgis.html.
14. Hammer, D. A,, T.J. Wickham, M. L. Shuler, H. A. Wood and R. R. Granados,
1995. Fundamentals of baculovirus-insect cell attachment and infection, in
Baculovirus Expression Systems and Biopesticides, M. L. Shuler, ed., Wiley-
Liss, New York, NY, pp. 103-1 19,
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ecticides
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15. Hoover, K.,R. Herrmann, H. Moskowitz, B. C. Bonnlng, S. S. Duffey, B.F.


McCutchen and B. D. Hammock, 1996.Thepotential
of
recombinant
baculoviruses as enhanced bioinsecticides, Pestic. Outlook, June 1996,21-26.
16. Hostetter, D.L. and P. Putler,1991. A new broad-spectrumnucleo
polyhedrosis virus isolated from a celery looper Atlagrapha falctfera (Kirby)
(Lepidoptera: Noctuidae), Envirorr. Entonrol., 20, 1480-1488.
17. Jacob, T. K.,1996.Introduction and establishment of baculoviruses for the
control of rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae) in
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18. Maeda, S., 1995. Further
development of recombinantbaculovirus
insecticides, C w r . Opirr. Biotechrrol., 6(3), 3 13-3 19.
19. Medugno, C. C., J. M. G. Ferraz, A. de H. N. Maia and C. C. L. Freitas, 1997.
Evaluation of a wettable powder formulation of the nuclear polyhedrosis virus
of Anticarsia gemnratalis (Lep.: Noctuidae), Pestic. Sci.,31, 153-156.
20. Menn, J. J., 1996.Biopesticides: has theirtimecome?, J. Grvworr. Scr.
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21. Moscardi, F., 1999. Assessment of the application of baculoviruses for control
of Lepidoptera, Annu. Rev. Etzfonrol.,44, 257-289.
22. ORcilly,D. R., and L.K. Miller,1991.Improvement of abaculovirus
pesticide by deletion of the egt gene, Bio/Tecllnology, 9, 1086-1089.
23. Park, E. J., C-M Yin and J. P. Burand,1996.Baculovirusreplicationalter
hormone-regulated host devclopment, J. Gen. Virol. 77(3), 547-554.
24. Pingel, R. L., and L. C. Lewis, 1997. Field
application of Bacillus
thuringierrsis and Atragrapha filcifera multiple nucleopolyhedro v m s against
the corn earworm (Lepidoptera: Noctuidae), J. Econ. Entotno/., 90(5), 1195-
1 199.
25. Possce, R. D., A. L. Barnett, R. E. Hawtin and L. A. King, 1997. Engineered
baculoviruses for pest control, Pestic. Sci., 3 I , 462-479.
26. Shapiro, M., 1995. Radiation protection and activity enhancement of viruses,
111 Biorational Pest Control Agents: Fornlulation and Delivery, F. R. Hall and
J. W. Barry, eds., American Chcmical Society, Washington, DC, pp. 153-164
27. Treacy, M. E., 1999. Recombinant baculoviruses, in Biopesticides: Use and
Delivery, F. R. Hall and J. J. Menn, eds., Humana Press, Totowa, N.J., pp. 321-
340.
28. Vail, P. V., D. F. Hoffmann and J. S. Tebbets, 1996. Effects of a fluorescent
brightnerontheactlvity of Atzagrapha falcifera (Lepidoptera:Noctuldae)
nuclear polyhedrosis virus to four noctuid pests, Biol. Control, 7(1), 121-1 25.
29. Van Beek, N. A.M.,and P. R. Hughes,1998.Theresponsetime of insect
larvae infected with recombinant baculoviruses, J. Irrverfebr. Pathol., 72, 338-
347.
30. Volkman, L. E., 1997.Nucleopolyhedrovirusinteractions with theirinsect
hosts, Adv. Vincs Res., 48, 3 13-348.
31. Wang, P. and R. R. Granados, 1997. An intestinal mucin is the target substrate
for a baculovirus enhancin, Proc. Nut. Acad. Sciences, 94( 13), 6977-6982.
32. Washburn, J. O., B.A. Kirkpatrick, E. Haas-Stapletonand L. E. Volkman,
1998.Evidencethatstilbene-derivedopticalbrightener, M2R, enhances
Autograph californica M Nucleopolyhedrovirus infection of Trichoplusia ni
166 Chapter 6

and Heliothis virescens by preventing sloughing of infected midgut epithelical


cells, Bioi. Control, 11 (I), 58-69..
33.
Weeden,
SheltnandHoffmann, 1998.
Baculoviruses,
Biological
control.
A
guide to natural enemies in North
America,
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Univ., http://
www.nysaes.cornell.edu/ent/biocontrol/pathogens~aculovi~ses.html.
34. Wood, H. A,, 1995.
Development and
testing of genetically
improved
baculovirus
insecticldes, in Baculovints
Expression
Systems and
Biopesticides, M.L. Shuler, ed., Wiley-Liss, New York, pp.91-102.
35.Wood, H. A. and P. R. Huges,1995.Development of noveldeliverystrategies
for use with genetically enhanced baculovirus pesticides, in Biorutional Pest
Control Agents: Formulation and Delivery. F. R. Hall and J. W. Barry, Eds.,
American Chemical Society, Washington, DC.
Biofungicides

1 Introduction
Increasing instances of resistance development and environmental concern are
the majordrivingforcesbehind the developmentsofbiofungicides for the
controlofplant diseases. Several scientific groupssearching for biological
means for control have addressed a number of significant fungal diseases. An
overview of the progress achievedin commercializing biofimgicides and control
of some economically important plant diseases is provided in this chapter.
Diseases of the plants are the result of the interaction of a pathogen, a
susceptiblehostplantand the environment.Incontrolof entities, biocontrol
agentsareused to target both the disease (a process)and the organism
(pathogen). However strategies for controlling the disease process (therapy) can
differ from that used to control the pathogens (C. L. Wilson, 1997). The plant
pathogens cause problems only undercertain environmental conditions although
most require free water in the environment to becomeactive (Figure 7.1).
Alllivingorganisms are subject to disease, parasites andpredators.
Each of these natural means of control can be exploited to protect crops from
pests. Microbial antagonism and hyperparasitism, phenomenon that exploit the
effects ofoneormoreorganisms inthe environmentagainst the pathogen,
comprise a viable means for control of plant diseases. Organisms that parasitize

167
168 Chapter 7

Certain
Plant
pathogen Interaction Susceptible under
environmental
organism)
(An with plant
host
conditions

results
target

target disease
Plant
Biofungicides _____)
(A process)

Figure 7.1 Blofungicldes target both plant diseaseand plant pathogen.

and destroy the pathogen, are applied before planting the crop.On the other
hand, organisms that function as antagonist by competing with the pathogen for
supply of nutrients or space; or inhibiting its growth by antibiosis where
antagonists secrete metabolites harmful to pathogens, are applied at the time of
planting. Antagonistic microorganisms that compete or directlyattack the
pathogen are mixed with the soil, added to furrows, used as seed treatment and
sprayed into foliage or fruit. A number of reviews on biological control of plant
pathogens and plant diseases have appeared in the literature (Be'langer et al.,
1998, Chalutzand Droby, 1998; Backman et al., 1997; El-Ghauth, 1997;
Mehrotra et al., 1997; Whipps, 1997; Utkhede, 1996; Malony, 1995 and Cook,
1993).
Like other disease management methods, biocontrolideally,aimsto
suppress disease sufficiently so that avoidable yield losses are minimized and
crop quality is maintained at an acceptable level. Biological systems however,
tend to be unreliable due to the effect of widely varying environmental
influences on their efficacy. For this reason, biofungicideshavebeen more
successful with protected crops and post-harvest infestation control, where there
is a degree of control on environmental parameters. Biofungicides complement
synthetic fungicides for the control of disease on agronomic and horticultural
crops.Therefore, they are preferred for integrated use incombination with
fungicides (Figure 7.2).
Commercial biofungicides are characterized for their consistent
suppression of fungal pathogens under field conditions, and easy mass
production in standard fermentation facilities. These can be classified into three
categories based on their target applications: soilborne pathogens, foliar diseases
and post-harvest rots during storage. All the three categories have been
extensively studied and are reviewed in the following paragraphs. Some of the
important soil-borneand post-harvest diseases have also been discussed. A
discussion on two important biocontrol agents, one a fungal and another a
Biofungicides 169

/ control
agents

preferred
succesful
Protected crops
(Green
houses)
Integrated
use due to Wdely varying
environmental
fungicldes
with
post-harvest
and
on control feld crops
influences

Figure 7.2 Biologicalcontrol is more reliable in protectedcrops.

bacterial system has been included. The production and formulation aspects of
bacterial andfungalbiofungicideshaverequiredconsiderabledevelopmental
efforts and these aspects havealso been reviewed here.

2 Biological Control of Soil-Borne Diseases


In the field, many ineradicable soil-borne diseases occur suchas those caused by
fungal plant pathogen species of Fusarium, Pythium, Verticillium, Rhizoctonia,
Macrophomina, Phytophthora and others, which cause either seed rot before
germination or seedlingrot after germination. The diseases are often termed pre-
andpost-emergencedamping-off,orseedling blights. Thesoil-borneplant
pathogens cause major economic losses in agriculture. The present methods for
control of these diseasesare inadequate and also cause non-target side effects on
beneficial microorganismsand the environment.Thus, there is aneed for a
suitable alternative to synthetic pesticides. Alternative disease
control is
sometimespossiblethroughdevelopmentofcrop plants that are resistant to
disease. Unfortunately, resistant plants are notavailable for manydiseases
caused by soil-borne plant pathogens.
Biologicalcontrol is anothermeansofcontrollingsoil-bornefungal
disease, which is presently receiving muchattention. Biological control has been
more successful in the plant rhizosphere employing both bacterial and fungal
biocontrol agents, than in the phyllosphere.
170 Chapter 7

2.1 Bacterialand ActinomycetousBiofungicides


for Soil-borne Pests

A listofcommercialbacterialbiofungicidesforcontrol of soil-borneplant
diseases is given in Table7.1.

Table 7.1 Commercial bacterial biofungicldes for control of soil-borne


plant diseases.

Microbial Target
Trade Mamrfacfurer DiseasesKrops
Name
Antagonist Pathogen

Bacillus
subtilis Kodiak Gustafson, Iythium Seedling diseases
(Strain GB03) Inc., Plano, ultimrrnl, and late season
TX, USA Rhizoctonia Rhizoctonia root rot
solarli and in cotton
Fusarium spp

B. subtilis Epic Seedling diseases


(Stram GB07) of cotton and
legumes

Bacillussubtilis Serenade AgraQuest, Powdery and


(Strain QST7 13) Davis, CA, downy mildew, late
USA and early blight and
brown rot

Agrobacferiunr Norback New Bio Agrobacferiunr Preventive (not


radiobacter 84-C Products, tunlefaciens curative) control of
(Strain K 84) Corvallis, OR, crown gall in
USA grapes, blueberries,
bushberries,
Galltrol AgBioChem, deciduous trees and
Orinda, CA, ornamental plants
USA

A. radiobacfer NoGall Bio-Care Also prevents


(Strain K 1026) Technology, transfer of agrocin
Somersby, resistance to
Australia A. tunlafaciens
Biofungicides 171

Microbial Trade Target


Antagonist Name Marllfictltrer Pathogen DiseasesKrops

Enterobacter Phytophthora Crown and root rot


aerogetles cactorunr of fruit trees (e.g.
(Stram B 8) apple) by soil and
trunk drench

Pseudomonas Dagger G Ecogen, Pythiurn Seedling diseases


jluorescens Langhorne ultirnunr. of cotton
PA, USA R.. solani

P jluoresccns Conquer Mauri Foods, Pseudomonas Bacterial blotch of


NCIB North Ryde, tolaasil mushroom
Australia

P jluorescens Victus Sylvan Spawn, P. tolaasil Bacterial blotch of


NCIB 12089 Kittanning, PA mushroom

P. jluorescells BioCoat S & G Seeds, Fusarium wilt in


wcs374 The radish and
Netherlands carnation

Bttrkholderia Deny Stine Fusarium spp. Disease caused


cepacia (formerly Microbials, Pytl1ium spp. lesion, spiral, lance
(Wisconsm Blue Haskins, KS, and and sting
M36) Circle) USA Rhizoctonia nematodes
solani

B. cepacia Leone Dominion BOttyliS. Bottytis, and


(Stram 679-2) Biosciences, Septoria and Phytoplrthora on
Walnut Creck, Plrytopkthora, potatoes and
CA, USA SPP. Septoria on wheat

Streptotnyces Mycostop Kemira Agro Fusarium oxy- Wilt of carnation;


griseovirtdis Oy, Helsmki, sporzcnr f. sp. root diseases and
K-6 1 Finland rliantlrr and foot rot of
Alterrraria cucumber,
AgBio brassicrcola Fxsariztnr disease in
development, and Plryto- wheat and in
Westmmster, plrthora, tropical plantation-
CO, USA Thielaviopsrs crops; damping off
and Macro- diseases in cauli-
pholnina spp. flower, broccoli,
cabbage and
ornamental plants.
172 Chapter 7

Brannen and Kenney (1 997)of Gustafson have reported successful use


of Kodiak@,a Bacillus subtilis strain G B 0 3, as a bacterial seed treatment for
control of seedling diseases of cotton, beans and othercrops. Kodiak@ is
reported to be effective against pathogenic species of Rhizoctonia and Fusarium.
The product is formulated as wettable powder and is compatible with several
seed treatment fungicides. Similarly, Bacillus subtilis strain A13 is inhibitory in
vitro to several plant pathogens and is being marketed under the name Quantum
4000' for peanuts and Epic@for seedling diseases in cotton for the control of
Fusarium spp.and Rhizoctonia spp. (Utkhede, 1996).A B.subtilis strain
QST7 13 hasbeen found to be effective in controlling various foliarplant
pathogens, such as Botlytis, powdery mildews, fire blightandsomedowny
mildews. AgraQuest is marketing this strain as Serenade@. Another biofungicide
based on a B. pumilus strain has been in field trials and is expectedtobe
available in the market in 2000 under the trade name Sonata (Marrone, 1999). B.
cereus UW85 is another gram-positive bacterium that has been found effective
in controlling Phytophthora damping off and root rot of soybeans under diverse
field conditions(Emmert and Handelsman, 1999). Presently, the greatest
commercial usage of rhizosphere-colonizing bacterial biocontrol agents, largely
Bacillus spp. are in combination with seed dressing chemical fungicides. The
fungicides are still necessary because Bacillus spp. are too slow in activating
and effectively colonizing the seed and emerging root to deal with fast-growing
pathogens. Dicarboximides such as vinclozolin and iprodion are best suited for
use along with Bacillus biocontrol agents (Seddon et al., 1995). The marketing
strategy has been to address season-long root health, reducing fungal pathogen
damage caused after the seed-treatment fungicides have dissipated,usually
occurring more than two weeks after planting.
Agrobacterium radiobacter strain K 84is another example of economic
biologicalcontrol with a bacterial antagonist.Thecommercialpreparation
Galltrol@or Norback 84-C@is reported to be effective in controlling crown gall
(A.tumefaciens) on stone fruits, commonly attributed to their production of
agrocin, an antobiotic. A recombinant A . radiobacter (derivative K 1026) was
engineered to prevent transfer of agrocin resistance, from the bacteria A .
radiobacter to A . tumefaciens (Jones et al., 1989; Maloney, 1995). The
engineered strain was marketed in Australia in 1988 under the trade name
NoGall@..Yetanother bacterial antagonist Erzterobacteraerogenes strain B8
reportedly controlled crown and root rot of fruit trees caused by Phytophthora
cactorum (Utkhede, 1996).When E. aerogenes (B8) was alternated with
metalaxyl, P. cactorum infection was significantly reduced and fruits yield of
apple trees increased.
Ecogen's Dagger G@,a Pseudomonas fluorescens granular peat-based
formulation controls seedling pathogens of cotton. Similarly,
another
commercial preparation of P. fluorescens, Conquer@ has been found effective
Biofungicides 173

forcontrolof Pseudomonas tolassil onmushrooms in Australia. A P.


fluorescens WCS374preparationBioCoat@ is applied as aseedcoating to
radish and carnationfor control of Fusarium.
Burkholderia cepacia, a bacterium formerly known as Pseudomonas
cepacia was renamed in honor of Walter Burkholder, aCornel1 University plant
pathologist, who first reported it to be the cause of sour skin of onions. It has
been shown to be an effective biocontrol agent against soil-borne as well as
foliar and post-harvest diseases. Many strains of B. cepacia produce one or more
antibiotics active againstabroadrangeofplantpathogenic fungi. Deny@.is
registered with US.EPA for use as microbial biofungicide.
KemiraAgroOyofFinlandhasput in the marketafreeze-dried
actinomycete product Mycostop@ based on Streptomyces griseoviridis. It is a
wettable powder containing mycelium and spores and can be applied to seed as
a dry powder or suspended in water and used as a dip, spray or drench. It is
effective in controlling wilt diseases of carnation, root and foot rot of cucumber,
fusaria1 diseases in wheat and in tropical plantation crops, damping-off diseases
in cauliflower, broccoli, cabbage and ornamentalplants.

2.2 Fungal biofungicides f o r soil-borne pests

Thefungalorganismsresearched as biocontrolagentsareprimarily
filamentous fungi, such as, Gliocladiumvirens and Trichodermaharzianum.
However, there are also examples of someyeast-like fungi such as Ampelomyces
quisqualis. Theenvironmentalconditionssuch as temperatureandmoisture
affect the growth and survival of fungal biofungicides and limit their efficacy.
The application of these biofungicidesis for control of root-infecting pathogens,
e.g.. Pythium,Rhizoctonia and foliar fungalpathogens, e.g. powdery mildew
and Botlytis. Various mycofungicides commercialized for control of soil-borne
plant diseases are tabulatedin Table 7.2.
Cornel1 Universityresearchershaveproducedahybrid strain ofa
filamentousfungus, Trichodermaharzianum 1295-22, byprotoplastfusion
between T. harzianum strains T-12andT-95. It hasefficacyagainstawide
range of plant pathogenic fungi including, B. cinerea, protects the root system
against Fusarium, Pythium and Rhizoctonia onanumberofcropsincluding
corn, soybeans, potatoes, tomatoes, beans, cotton, peanuts, turf, trees, shrubs and
other transplants andornamental crops. It is compatiblewithmanystandard
chemical seed treatments (e.g. Captan) and is applied directly over chemically
treated seedsat the timeofplanting(Sivanet al., 1991).Twocommercial
productsRootShield@andBio-Trek22G@are registered byBioWorks, Inc.,
Geneva, NY. There is another product
being
developedby
Mycotech
Corporation is a blend of two complementary strains of Trichoderma under the
trade name EcoGardTM.It has been foundeffective against various soil borne
174 Chapter 7

Table 7.2 Commercial mycofungicides for control of soil-borne plant diseases

Fttngal
Pathogetr
Mamcfactroer
Pathogen
Target DiseaseKrops

Fusarirrnl Fusaclean
Natural
Plant oxysponrnr
F Fusarium wilt of
oxyspol7rnrflower and vegetable
Protection,
(Nonpathogenic crops
Nogueres, house
in green
strain Fo47) France

Gliocladirrnr SoilGard
Thermo Pythiunr spp.,
Damping
and
off
virens GL2 1 Trilogy, Rhizoctonia root rot of
Columbia, solarti and ornamental and food
MD Sclerotircnr crop plants in green
rolfsi house.

Trrclrodertrra RootShield
BioWorks,. Pytytkiunr spp.,
Damping off and
hnrzranunl and T-22Geneva,
NY, Rhrzoctorroa root
rot
of
corn,
bean
(Strain 1295-22) Planter USA solarri and and vegetables
Box Sclerotirtra
honloeocarpa

Trichoderttla Promote JH Blotech, Pythiunr, Greenhouse


crops,
harzranrtnl and Ventura,
CA, Rhrzoctonia nursery
transplants
T vrrrde USA and Fusariunl seedlings
and

T. Irarzrarrurrl Binab
Bio-lnno-
T Chottdrostereum Tree
wood
decays,
(ATCC 20476) vatlon
AB, pwpureton, silver
leaf
plum
of
and Algaras, Endothia Chestnut
apple;
and
T polyspol-ul?l Sweden parasitica, pinewood
blight,
root
(ATCC 20475 Verticillittnl decay
nlalthousei,
Heterobasidiorr
atrnosrm,
Artrlillarra
nrellea and
Ceratocystis
rhi.

Pytlri~cnl Vyskumny
Poly- P. ultinlrcnl Dampmg-off
of
sugar oligarrdncnr
ustav, gandron
Piestany,
Slovak
Biofungicides 175

Peniophora RotStop
Kemira
Agro Stem and root rot of
(I'hlehia) flelsinki. pinc
giganlea Finland

P.g. Ecological
suspension Labs., Dover.
UK
.Ispergill2t.s Kaliscna IAlil. New Cereals. millcts.
niger AN21 India Dclhi. pulses. oil-seeds.
fruits. tubers.
vegetables and
ornamentals

diseases and also gives goodcontrol of Botrytis on strawberries (Mycotech,


1999). Similarly, a T. harzic~numproduct hasbeen reported to be effective
against Sclerotium rol/.iii in chick pea (Mukhopadhyay et al., 1994).
SoilGard'" (Therm0 Trilogy)developed by Biocontrol of Plant Diseases
Laboratory (BPDL) of USDA and Grace Biopesticides, is a granular formulation
for control of soilborne plant pathogens, including Pythium spp., Rhizoctonia
soluni. Sclerotium rol/sii and others by the common soil saprophytic fungus,
G'/ioC/UdiZlr?7virens GL21. It produces an antibiotic gliotoxin that is implicated
as thekey factor in its biocontrol activity against Pythizrm zrltimum and
Rhizoctonia solani (Wilhite et al., 1994). Phlehiu gigantea was one of the first
microbes usedfor control of Heterohasidion annosum in trees. Since the
pathogen can colonize freshly cut stumps and then spread through root grafts,
the product Rotstop@is applied to freshly cut stumps.
A few Pythium species are known to be aggressive mycoparasites.
Pyyrhium nun, attacks Pythiumultimum and P. vexans, by coiling around the
hyphae of the host, forming appressoria-like structures and then parasitizing the
hyphae (Lifshitz et al., 1984). Similarly It is reported that sugarbeet seeds coated
with oospores of P. oligandrum effectively controlled preemergence damping-
off caused by P. ultimum (Martin and Hancock, 1987). Pythium oligandrum is
an aggressive mycoparasie andcanbeused as a biological control agent of
several plant pathogenic fungi. A videomicroscopic study depicts how Pythium
oligandrum recognises its host fungi and destroys them (Figure 7.3) (Laing and
Deacon, 1991). A product based on P. oligandrum has been commercialized in
the trade name Polygandron" by Vyskumny ustav of Slovak Republic.
176 Chapter 7

Figure 7.3 Videotaped interaction between Pythium oligandrum (Po) and the plant
pathogenic fungus Botrytis cinerea (Bc) on- a thin fiim .of water agar.
(Curtsey: J. W. Deacon). (a and b) The hyphal tip of Bc contacted the
hypha of Po, (c) The tip of Bc suddenly lysed (d and e) expelling the
protoplasm, (f) Po produced many hyphal branches using the spilled
protoplasm as a nutrient source

Kalisena@,aproductbasedon Aspergillusniger isolate AN27 is


reported to control several soil borne pathogens including F. oxysporium, F.
solani, Pythium spp., R. solani and S. sclerotiorum in diverse group of crop
plants(Senet al., 1996). Theproductisreprtedtobe in earlystages of
commercialization.

3 Soil-borne Diseases
Some of the economically devastating soil borne diseases, such as wilt,
Fusarium
Phytopthora, Sclerotiniaand Pythium diseases and their control are discussed in
following paragraphs.
Biofungicides 177

3.1 Fusarium wilts

Fusarium wilt and fusarium root-rots are among the most severe plant diseases
caused by pathogenic strains of Fusarium oxysporum, one of the most common
species among soil fungi in cultivated soil. These fungi have a large diversity of
strains, all of them being saprophytic; many of them are parasitic and some of
them pathogenic and induce either root rot or tracheomycosis. Fusarium prefers
warm, dry conditions typical of summer. The wilt inducing F. oxysporum strains
show a high degree of host-specificity; and certain saprophytic strains of the
same have been tried for reducing the pathogenic population.
Theconcept of certain soils being conducive or suppressive to
disease refers to the soils that favor the expression of disease, or to the soils that
are inhospitable to some plant pathogens respectively. In suppresivesoils,a
pathogen cannot become established or if established cannot initiate disease.
Presence of a large population of non-pathogenic F.oxysporum in soil,
suppresses fusarium wilts. The modes of action include a competition between
the pathogenic and non-pathogenic F.oxysporum for nutrients and infection
sites in the rhizosphere and also induction of resistance in the plant (Figure 7.4).

Ll- 4r
Fluorescent Pseudomonas spp. are among the most abundant bacteria
in the rhizosphere. They are characterized by the production of yellow-green
pigments(pyoverdines).These fluoresce under UV lightandfunctionas
siderophores. It has been established that some strains of fluorescent
Pseudomonas spp. are able to inhibit the growth or activity of fungal pathogens
including F. oxysporum.The main mechanism of the antagonistic expression by

Fluorescent
Non-pathogenic su resses Fusarium
F. oxysporum
insoil wilts P s ~ ~ ~

due to due to
competition
competition
between

IronPathogenic
and
non-pathogenic
F. oxysporum spp. Required

Spore germination,
Nutrients and penetration
and
infection sites
infection

Figure 7.4 Competition mechanisms of suppression of Fusarium wilts


178 Chapter 7

fluorescent Pseudomonas spp. against F. oxysporum is the competition for iron


(Figure 7.4). It has been well established that fluorescent Pseudomonas spp.
producing the catechol-hydroximate sidorophores are very efficient competitors
for iron (Fe3') required for germination, penetration and infection (Baker and
Griffin, 1995). Reducing availability of iron to pathogenic F. oxysporum results
in an increased level of soil suppressiveness. Additionally, it is also likely that
fluorescent Pseudomonas mediated induction of systemic resistance is also
involved in the reduction of disease.
Lemanceau et. al. (1992,1993) demonstrated that acomplementary
effect of non-pathogenic F.oxysporum andfluorescent Pseudomonas spp.
significantly contributed to the suppressiveness of soils to Fusarium wilt. They
used a siderophore-deficient mutant of P. putida to demonstrate that competition
for iron, resulting from the activity of the bacterial strain, controlled the efficacy
of competition for carbon between strains of F. oxysporum.
The efficacy of biological control depends on the population density of
the antagonistic microorganisms, but in case of F.oxysporum the ratio of
population density (> 10) of the non-pathogenic versus the pathogen is more
important than the absolute value of the population density itself (Alabouvette et
al., 1998). Specific isolates of non-pathogenic F. oxysporum have been reported
to be highly effective at controlling Fusarium wilt of tomato (50430% reduction
of disease incidence) and other crops (BARC, 1999). These results indicate that
non-pathogenic Fusarium spp. have potential for further developmentas
biological control agents and canbea viable alternative to fumigation with
methyl bromide for severalcrops. Fusaclean@, commercially
a available
microgranular product based on nonpathogenic Fusarium oxysporum controls
pathogenic F. oxysporum and F. moniliforme on basil, carnation, cyclamen and
tomato (Table 7.2) (Fravel et al., 1998).

3.2 Phytophtltora
Diseases

Fungi in the genus Phytophthora cause important diseases on many


economically important crops worldwide. The late blight pathogen P. infestans
causes serious losses of potato crops. Phytophthora root, crown, and collar rots
arecommonand destructive diseases of fruit trees. Disease development is
favored by cool (15 to 25"C), wet, humid conditions and is most severe in poorly
drained sites. Significant damage occurs only when soils are extremely wet or
saturated. Phytophthora grows through the root destroying the tissue, which is
then unable to absorb water and nutrients.
Many bacteria, actinomycetes and fungi are antagonistic to
Phytophthora, and undoubtedly such systems are operative in nature. However,
the development of effective biological control of Phytophthora specieshas
been fraught with difficulties because of their ability to produce several formsof
Biofungicides 179

inoculum (zoospores, sprangia, chlamydospores, oospores and


mycelium)
rapidly and repeatedly, their ability to penetrate and infect a host plant within a
few hours, their propensity to exist in soil in depths that allow them to escape
most antagonists and in some cases their wide host range (Erwin and Riberio,
1996).
Crown and root rot of fruit trees is primarily caused by Phytophthora
cactorum (Utkhede, 1986). Several fungi show promise for biological control of
this pathogen by parasitizing and lysing its propagules in soil. For instance,
Trichoderma and Gliocladium species suppressed P. cactorum onapple
seedlings in glass house tests. A cornmerical preparation Binab TO, containing
spores of Trichoderma spp. inhibited formation of sporangia and caused lysis of
P. cactorum mycelium in vitro (Table 7.2). Binab T@,reduced the size of lesions
(54%-57%) on inoculated apple twigs as well as on apple trees sprayed with it
(Figure 7.5) (Orlikowski and Schmidle, 1985).
Although Trichoderma species antagonise Phytophthora spp., isolates
of this fungus also have been shown to stimulate the formation of oospores in
normally self-sterile isolates of Phytophthora. Response of T. viride on P.
cinnamomi is reported to be both lysis of hyphae and induction of oospores
(Reeves, 1975). However, production of these resting stores may compromise
the effectiveness of biological control.
There is much evidence to demonstrate that most Phytophthora spp. are
relatively non-saprophytic anddo not grow well incompetition with other
microorganisms in soil.Simply filling in the transplanting holes with non-
infested soil controlled the seedling disease of papaya caused by P. palmivora.
The practice exploited the inability of P. palmivora to compete with naturally
occurring microorganisms in soil (KO, 1982).

Phyfophthora Parasitizing and lysing


Trichodermaand su
Gliocladium spp.
E-cactorum on by Phytophthora propargules
apple seedling in soil

Binab -$
(T' hapianurn and
T. polysporum)

reduce
- inhibits Formation of sporangia
andsurvival of mycelium
of Phyfophthora cactorum

Size of lesions on
(-55%) Apple twigsas
well as trees

Figure 7.5 Mechanism of biologicalcontrol of Phyfophthoru disease in apple


seedlings.
180 Chapter 7

3.3 Sclerotinia Diseases

As a group, Sclerotinia spp.are fungi, typically comprised of soil-borne


pathogens that produce monocyclic diseases the
in
rhizosphere
and
phyllosphere. Diseases caused by Sclerotinia spp. are difficult to manage
because of their widespread distribution, sporadicoccurrenceandlong-term
survival in soil. There are three economically important Sclerotinia species viz.
S. sclerotiorum, S. trifoliorum and S. nlinor.
Sclerotininsclerotioruln is the most widespread and economically
important pathogen within the genus Sclerotinia because of its widespread
distribution and host range. It is considered an opportunisticinvader of
senescent or dead foliar and floral tissues. Theepidemiology of disease is
primarily associated with carpogenically germinated sclerotia that produce
apothecia which release ascospores. Infection of S. trifoliorum also typically
originates from ascospores, but germinating spores penetrate foliage directly and
establish quiescent infections, eventually becoming more aggressive and
developing into an expansive leaf and stem rot. On the other hand, S. minor is
associated with eruptive or myceliogenic germination of sclerotia in the
rhizosphere, which results in direct infection of roots and crowns of susceptible
plants.
The strategy adopted for biological control of diseasescaused by
Sclerotinin spp. is to reduce the concentration of initial inoculum by killing
sclerotia or inhibiting their germination. Use of parasitic fungi or mycoparasites,
have been found useful in reducing the number of sclerotia in infected soils
(Figure 7.6).

Mycoparasites
Sclervtinia spp. treated with such as
infected s o i l Sp. sclerotivorum

Killing sclerotia
by
I
Y
Number of
reduce

+
or inhibiting their 4
sclerotia
germination
I
control

Diseases caused
by Sderotinia spp.

Figure 7.6 Mechanism of biologlcal control of diseases caused by Sclerotirzia spp.


Biofungicides 181

One of the most effective mycoparasites is Sporidesmium


sclerotivorum that behaves as an obligateparasite on sclerotia of Sclerotinia spp.
After sporulating on the surface of the sclerotium, Sp. sclerotivorum can grow
for upto 3 cm through soil to infect anewsclerotium.The ability of Sp.
sclerotivorum to proliferate andgrowthrough soil to infect new sclerotia,
facilitates the epidemic destruction of sclerotia, regardless of whether the host
plant is present or not (Fravel, 1998). In order to reduce the number of viable
sclerotia in thesubsequent crops, applicationofsporesuspensionsof Sp.
sclerotivorum to the phyllosphere successfully controlled Sclerotinia diseases
that do not involveascospores in the disease cycle. Thisapproachhasbeen
successfully used to control lettuce drop incited by S. minor (Zhou and Boland,
1998)
There are eight other fungi reported, that are antagonistic to Sclerotinia
spp. These are Teratosperma oligocladium,
Coniothyrium
minitans,
Dictyosporium elegans, Penicillium citrinum, Talaromyces flavus, Gliocladium
virens, Trichoderma spp. and Elwina herbicola.

3.4 Pytlrium Diseaes

Species of Pythium are best known as pathogens of crop plants. They cause
damping-off and other seedlingdiseases, and also progressively destroy the root
tips ofolder plants, leading to decline diseasesoforchard trees andother
perennial crops. It belongs to a group of fungi called the water molds. As the
name implies, thesefungi flourish in anenvironment in whichwater is
constantly available. Pythium flourishes when soils are cool and wet, such as
during the spring. Itcan infect seedswithin 12 h. of planting. Sporangia of
Pythium ultimum must germinate in order to incite seed and seedling rots. This
germination occurs within 0.5-1 hr after exposure to exudates of germinating
seeds and colonizes the seed very rapidly after planting (Harman and Nelson,
1994).Incottonandpossiblyotherplant species, fatty acidsareimportant
germinationstimulation
molecules,
with linoleic acid
being the most
stimulating. A biocontrol agent capable of inactivating the stimulatory activity
of seed exudates and linoleic acid, would control damping off rot of seeds and
seedling.

4 Biological Control of Aerial Plant Diseases


The availability ofseveral relatively effective fungicides for useagainsta
majorityof foliar fungalpathogens,hasbeenadisincentivetodevelop
biocontrol agents for these pathogens. However, the now frequent occurrence of
fungicide resistance, for example, to the benzimidazolesanddicarboximides,
182 Chapter 7

has necessitated the development of alternate strategies. Particularly, biocontrol


of Botrytis cinerea that causes the disease grey mold and Uncinula necator and
Sphaerothecafuliginea causing pathogens of powderymildewshas been
studied.

4.1 Grey mold disease

The necrotrophic Botrytiscinerea causes the disease grey mold which is a


serious economic problem in a number of fruit crops, such as grapes, strawberry,
raspberry, orchard fruits and green house cropssuchastomatoes and potted
plants. Botrytis cinerea, a deuteromycete fungus is an important plant pathogen
with an exceptionally broad host range. Conidia of B.cinerea, the primary
inoculum source, first attach to substrata by a hydophobic interaction that is
easily disrupted and then, upon germination, attach strongly, through secretion
of an extracellular matrix by germ tubes and appresseria but not the conidia
themselves, known as f h g a l sheath. The enzymatic action occurs immediately
after the pathogen comes into contact with the host and continues through the
time of penetration. It typically requires exogenous nutrients during germination
andgerm tube elongation. These pathogens are subjected to competition for
these nutrients with the indigenous saprophytic microbial community on foliar
surfaces (M. Wilson, 1997).
Suppression of sporulation of B. cinerea has been effectively achieved
through foliar applications of the saprophytic fungi Trichoderma spp.,
Penicillium spp. and Gliocladium roseurn in various hosts, including strawberry,
grapes and cucumber. Trichodertna harzianurn isolate T-39(Trichodex@) is
commercially available for control of greymold of grapes (Table 7.3).

4.2 Powdery MildewsDisease

Biotrophic pathogens causing powdery mildews, rust anddownymildews are


economicallysignificant. Particularly, powdery mildew is asignificant fungal
disease affecting many crops. Special structuresof the mildew penetrate the plant
epidermal cells and feed on cellular tissue, causing dwarfing of the plant and the
fruit, and cosmetic damage which is particularly undesirable on fresh produce.
Plants severely infected with these pathogens have reduced yields.Powdery
mildews such as U. necator and S. fuliginea are obligate parasites and typically
do not require exogenous nutrients during germination. This precludes the use of
nutrient competition as a biocontrol strategy, as used against B. cinerea. Instead,
hyperparasitism as an approach has been successfully applied in biocontrol of
powdery mildews on various plant hosts.
Biofungicides 183

Table 7.3 Biocontrolagents of aerial diseases.

Trade Microbial
Antagomsf Manufacturer Target Pathogen Confro1

Pseudolnonas
Blight
Ban Plant Health Erwinia Fire blight and
fluorescens A 506 Technologies,
arnylovora frost injury in
(Strain A 506) Boise, ID,USA pear and apple

Atnpelornyces AQ I O Ecogen, Uncinula necator, Powdery


mildew
quisqualis Langhome, PA, Etysiphe cichora in apples,
grapes
(Strain M IO) U.S.A. cearunr and chillis

Trichoderma
Trlchodex Makhteshlm Bottytis cinerea, Greymoldsl
hanianunl Chemical Works, Unclnula necator powdery
mildew
(Strain T 39) Sheva,
Beer Etysiphe cichora- in grapes,
Israel cinerea strawberries and
cucumber

Mixture of Grey Gold Eden Biosclence,


B. cinerea
7: lramatrmnl f Poulsbo, WA,
Rhodoforula U.S.A.
glufinis +- B.
nlegateriunl

A hngal hyperparasite Ampelornyces quisqualis has been identified


that infects most, if not all types of powdery mildews harmful to agricultural
products. It infects and forms pycnidia (fruiting bodies) within powdery mildew
hyphae. Thisparasitism reduces growth and may eventuallykill the mildew
colony. It reduces or eliminates applications of sulphur and DM1 fungicides such
as mycobutanil and fenarimol. Ecogen Inc. has commercialized an isolate "10
of A . quisqualis (AQ10@),that hasbeenfound effective on grapepowdery
mildew caused by U. necator and is also reported to give goodcontrol of
powdery mildew of cucumber at moderate disease pressure.
Verticillium lecanii, on the other hand is a polyphagous organism that
attacks the structure of its hosts and parasitizes powdery mildew fungi and rusts.
V. lecanii is reported to adequately control cucumber powdery mildew on a
resistant cultivar,although requirement of high humidity remains a limiting
factor in its efficacy (Verhaar et al., 1993).
Sporothrixflocculosa is a yeast like epiphytic fungus, that is a powerful
antagonist of powdery mildews. It was found effective in controlling cucumber,
rose and wheat powdery mildews. Temperature and humidity requirements are
important constraints in maintaining the efficacy of S. flocculosa. It induced a
184 Chapter 7

rapid plasmolysis of powdery mildew pathogen cells, suggesting its mode of


action by antibiosis.Some of the biocontrol agentscommercialized for the
control of aerial plant diseases are given in Table 7.3.
Effective biocontrol agents for control of serious foliar diseases such as
grape downy mildew, potato late blight, wheat powdery mildew and apple scab
remain to be identified (Froyed, 1996). However, Fusarium proliforatum was
recently proposed for downy mildew control on grapes (Hofstein and Chapple,
1999).

5 Control of Post-Harvest Decay of


Fruits and Vegetables
Fruits and vegetables are highly perishable while still maintainingactive
metabolism long after harvest. Post-harvest infection can occur either prior to
harvest or during the harvesting and subsequent handling and storage. In
general, most harvested commodities are resistant to fungal infectionduring
their early post-harvest phase. However, during ripening and senescence, they
become more susceptible to infection. Key infection sites for post-harvest
diseases are wounds created by harvesting and handling, though some pathogens
may directly penetrate. Post-harvest losses can be severe, if adequate handling
and refrigerated controlled atmosphere storage facilities are lacking.
Several biological control approaches, including the use of antagonistic
microorganisms, natural fungicides and induced resistance have been shown to
have apotentialasa antimicrobial preservative for harvested commodities.
Development of antagonistic microorganisms has been the most studied, among
these alternatives. Antagonistic microorganisms are selected for their ability to
directly parasitize and compete with plant pathogens, as well as to induce
resistance responses in the host to the disease. For enhanced biocontrol activity,
antagonistic microorganisms are combined with natural fungicides suchas
chitosan, which has capability of turning on host defenses to disease (C. L.
Wilson, 1997).
The chances of success of biological control measures are high in the
post-harvest environment.The partially controlled environment, particularly
temperature and humidity may help shift the balance of the interactions between
the host, the pathogen and the antagonist in favor of the antagonist.It is
generally assumed that the mode of action of biocontrol of post-harvest diseases
involve a complex mechanism, which comprises one or more of the following
processes: antibiosis, nutrient competition, site exclusion, induced host
resistance and direct interactions between the antagonist and the pathogen.
Many reportsdescribe the inhibition of post-harvestdiseases by
antibiotic producing microorganisms, based on the in vitro test results, in which
the antagonist inhibits the growth of the pathogen in culture. However, a debate
Biofungicides 185

hasensured, whether antibiotic-producingantagonistsshouldbeused on


fruitsand vegetables, resulting in introduction of antibiotics into our food. This
mayresultinresistancedevelopmentsinhumanstoantibioticsas well as
development of pathogen resistance (Kohl and Fokkema, 1998).
A number of antagonistic microorganismshavebeenidentifiedand
shown to be effective against various post-harvest pathogens on a variety of
harvestedcommodities(Table 7.4). Theseincludeapple,apricot,avocado,
banana,cabbage,carrot, celery, cherry, citrus,gerbera,grape,kiwi, litchi,
mango, nectarine, peach, pear, pineapple, plum, potato, roses, strawberry, sweet
potato, tomato, tulip bulbs and grains.
Utkhede(1996)hasreviewedsuccessfulapplication of antagonistic
bacterium Pseudomonas syringae (strain ESCll) for control of blue-mold rot
(Penicillium expansum), on wounded pear h i t as well as on Empire apples and
citrus fruits. P. syringe also provided control of greymold rot (Bottytis cinerea)
on Boscpears.EcoScience has commercialized this productunderthetrade
name BioSave 11@.

Table 7.4 Biocontrolagentsofpost-harvestdiseases of fruits and vcgctables

Disease/

Pseudomonas Bio-Save
EcoScience, Penrcilliurn Blue mold rot,
syringae 1IO Worcester, expansum,
P. green mold, grey
(ESC 1 I ) MA, U.S.A. digitatunl, Bottytis mold and mucor
cinerea and Mucor rot of apples
piriformis
P. syringae Bio-Save
EcoScience, Penicillium digitatunl, Green mold,
(ESC IO) 1000 Worcester, P. italicun~and blue-mold and
MA, U.S.A. Geotrrchunl candidurn sour rotof citrus
fruits

Bio-Save Blue mold and


100 grey mold in
pears

Burkholderia Deny
CCT Corp., Botrytis
cinerea Grey mold rot of
cepacla Carlsbad, apples and pears
(Wisconsin CA, U.S.A.
Is0 582)

Candida Ecogen,
Aspire Post-harvest
and spp
Bottytis
oleophila Langhorne, decay
Penicillium. spp. of citrus
1-1 82 apple and PA, U.S.A.
Chapter 7

Ecogen has introduced Aspire", a product based on a naturally


occurring yeast Candida oleophilu that is applied to disease-free fruit forcontrol
of post-harvestrot pathogens of citrus, pome fruits, grapes and apple by
protecting surface wounds frominvasionby pathogenic fungi. I t suppresses
decay formation via competition with germinating spores of prominent
pathogens (e.g. Penicillium spp.) for space and nutrients. The combination of
Aspire"with 200pdml thiabendazole oftenreducedthe incidence of decay,
caused by the green and blue molds (Penicilliumdigitaturn and P. italicum,
respectively), as effectively as conventional fungicide treatment. Furthermore,
Aspirewasfoundhigh efficacious against sour rot caused by Goetrichum
condidzrm, a decay not controlled by the conventional treatments (Droby et al.,
1998).
Cut flowers are often subjected to pathogen infections, during shipment
when high humidity has to be maintained. Even slight temperature changes can
lead to condensation of water on the surface of petals. Therefore, post-harvest
treatment of cut flowers is an attractive option for application of antagonists.
Redmondet al. (1987) foundtheyeast Exophiula jeanselmi very effective i n
preventing infections by B. cinerea on cut roses, and foundthat it gave
comparable results to ipridione in disease suppression (63%).

6 Biological Control with Trichoderma Species


One approach to biocontrol has been the use of antagonistic microorganisms that
compete with, or directly attack the pathogen. Saprophytic fungi in the genus
Trichoderma have been used to control a wide range of plant pathogenic fungi
responsible for the most important diseases suffered by crops of major economic
importance worldwide. In particular, isolates of Trichodermahurziunztm, T.
virens and T. humatunl havebeenusedwith success against soil-borne, seed-
borne diseases and diseases in the phyllosphere and against storage rots
(Tronsmo and Hjeljord, 1998). The mycoparasitic activity of Trichoderma spp.
is due to three antagonistic mechanisms that may be operative for the control
effects, including competition, antibiosis, production of cell wall-degrading
enzymes or a combination of these activities.
Mycoparasitism, the phenomenon of one fungus parasitizing another,
relies onthe
production of lytic enzymes by the mycoparasite for the
degradation of cell walls of the host fungus. Trichoderma, a mycoparasite, can
detect its host from a distance and, on detection, begins to branch in an atypical
way towards the pathogenic fungus. Itis speculated that trophic responseis
induced by nutrient gradients arising from the host ( I n addition, some strains of
Trichoderma may produce non-volatile antibiotics that inhibit and presumably,
predispose host hyphae infection
to before contact occurs). Following
recognition of its host, Trichoderma attaches to it and then either grows along
Biofungicides 187

Figure 7.7 Scanningelectronmicrographs of interactionsbetween Trichoderma


hanianum and R. Solani. (a) T. hanianum penetrates the hyphae of its
host and then exits @) T. hanumum (thin hypha) coils around R. solani
(thick hypha), there is a loss of turgor and marked cell lapse in R. solani
(Benhamou and Chet, 1993).

the host hyphae or coils around them (Figure 7.7). Secretion of lyhc enzymes,
chitinases and other hydrolasesfollow. Trichoderma produce appressorium like
bodies, which aid penetration of the host cell wall (Goldman et al., 1994).
Subsequent degenerative events in the host include disorganizationof cell wall
structure resulting in osmotic imbalances followed by intracellular disruption,
such as retractionofplasmamembraneandaggregation of thecytoplasm
(Figure 7.8).
The majority of pathogenic fungi contain chitin and 1, 3-p-glucans in
their cell walls, and dissolution of, or damage to, these structural polymers has
adverseeffectsonthegrowthanddifferentiation of suchfungi.Cell-wall-
degrading enzymes, particularly, chitinolytic enzymes produced by Trichoderma
188 Chapter 7

Recognition
Chemotropic of Secretion of
growth of __t the host by the ___t extracellular
Trichoderma mycoparasite enzymes

- -
Figure 7.8
Hyphae
penetration
Lysis of
the host

Mechanism of mycoparasitlcactivity of Trichoderma spp.

species, as also by closely related genus Gliocladium, can inhibit proliferation of


plant pathogenic fungi.
Chitinolyticenzymes have beenimplicated in biologicalcontrol
mechanisms. In in vitro experiments, it hasbeenshownthatchitinolyticand
gluconolytic enzymes isolated from T. harzianum inhibit spore germination and
hyphal(germtube)elongation in severalplantpathogens,including Botrytis
cinerea, Fusarium solani,F.graminearum,Ustilagoavenae and Uncinula
necator. However, Pythium spp., that doesnot have chitinasastructural
component of cell wall was not affected. The degree of inhibition was found
proportional to the level of chitin in the cell wall of target fungi. Combinations
ofthepurifiedenzymessuchasglucan1,3-P-glucosidasesandchitinolytic
enzymes resulted in a synergistic increase in antifungal activity (Tronsmo and
Hjeljord, 1998).
Zimand et al.(1 996) reported a new mechanism of biological control of
T. harzianum T39on Botlytiscinerea. Besidesthemhibitoryeffecton
germination and germ tube elongation of conidia of B. cinerea, T. harzianum T
39 reduced the production and activities of pathogenic pectolytic enzymes three
daysafterinoculation,resultingincreasedaccumulationofpecticenzyme
product (i.e. oligogalachuronides). These sugars can elicit the host plant defense
mechanisms, thus slowingthe disease severity.
Trichoderma harzianum has been successfully
applied in the
phyllosphere for the control or low disease incidence ofBotrytis cinerea, one of
the most serious pathogens of grapevines. However, the time of treatment is
importantforcontrol. Most effectiveprotectionisobtainedbytreatments
extending from the time of flowering to three weeks before harvest. The best
control is reported to be obtained in anintegratedprogramtogetherwitha
reduced dosage of fungicides. Even reduced amounts of the fungicide can stress
andweakenthepathogenandrender its propagulesmoresusceptible to
subsequent attack bythe antagonist.
Similarly, Trichoderma spp. have been used as seed coatings and as
granules for biological control of soil-borne pathogens. Control of Pythium spp.
Biofungicides 189

in tobacco,sugarbeetandcauliflowerhasbeenachievedby T. harzianum
through soil applications (Mukhopadhyay et al., 1986). As a seed protectant, its
efficacy may depend on the relative rates with which the antagonist and the
pathogen colonize the seed. However, here also integrated biological-chemical
seedtreatmentsmayprovide better results thantreatmentwith either the
chemical or biological agent alone. While the chemical protectant can be ideal
for short termprotectionof the seedor seedling, arhizosphere-competant
bioprotectantcancolonize the entire rootsystemandprovideaseasonlong
protection unattainable through acceptablelevels of chemical treatment.
Trichoderma biocontrol agents have also successfully controlled post-
harvest diseases. Carrots naturally infectedwith Mycocentrosporaacerina or
Rhizoctoniacarotae were treated with conidia of the cold tolerant isolate T.
harzianum P1 at the time of harvest and stored in the plastic-wrapped bins at 0-
5C. The cold tolerant Trichoderma isolate significantly reduced the number of
roots infected by both pathogens. On average, the amount of marketable crop
increasedby 47% after 6 months in cold storage, as compared to untreated
controls (Tronsmo, 1989).

7 Biological Control with


Fluorescent Pseudomonads
Fluorescent pseudomonads are the most frequently used plant growth promoting
rhzobacteria that functionbysuppressingdetrimentalrhizospheremicroflora
present in most soils. Pseudomonas fluorescens and P. putida suppress several
majorplantpathogens,especially the soil-borneones(Lahaet al., 1996).
Productionof
anti-fungal
metabolites,
such as antibiotics andhydrogen
cynamide and siderophore-mediated iron competition are primary mechanisms
bywhichthesebacteriasuppressdisease(Weller,1988).Siderophores are
biosynthetic compounds that are produced under iron-limiting conditions. They
serve to chelate the ferric ion (Fe3') from the environment into microbial cells.
Thus, iron availability is reduced for the pathogens.
Pseudomonas fluorescens 2-79 has been effective in control of 'take-
all' of wheat caused by Gaemannomyces graminis var. tritici, one of the most
economically-importantcrownandroot rot diseasesofwheatworldwide.
Aggressive root colonization is important for effective biocontrol with bacteria
and is positivelycorrelatedwith take-all suppression.The take-all decline, a
spontaneous diminution in disease and increase in yield that occurs after several
years of wheat monoculture is well known natural phenomenon associated with
the preponderance of
antagonistic
organisms
especially the fluorescent
Pseudomonas. The suppression of take-all of wheat depends on the ability of
bacteria to colonize the roots andproductionof an antibiotic phenazine-l-
carboxylicacid(PCA),asiderophore called pyoverdineandananti-fungal
190 Chapter 7

factor, 2,4-diacetyl phloroglucinol (ThomashowandWeller, 1990). However,


antibiotic rather than siderophore production has been suggested to the main
mode of action of P. fluorescens 2-79 in its control of G. graminis var. tritus
(Hamadan et al., 1991).
Fluorescent species of Pseudomonas species have been used to control
disease of various crops.Suppression of damping-off of cottoncaused by
Rhizoctoniusolani and Pythiumultitnum has been reported by applying P.
fluorescens isolated from the rhizosphere of cottonseedling. It hasbeen
suggested that sidorophores may not be the only key factors associated with
disease suppression. The antibiotics pyrrolnitrin and pyoluteorin produced by P.
fluorescens were the key antagonistic factors against R. solani and Pythium spp.
respectively. Suppression of several vescular wilts caused by Fusarium
oxysporutn, control of root rot of wheat caused by Pythium ultimum and potato
seed piece decay by Etwinia carotaova by Pseudomonas spp., are also reported.
Novartis AG has reported
development of genetically modified
Pseudotnonas strains with enhanced biocontrol activity against plant pathogenic
fungi such asspecies of Rhizoctonia and Pythium (Ligonetal.,1999). The
modified strains produce enhanced amounts of antifungal metabolites such as
pyrrolnitrin that are active against these fungal pathogens.

8 Production of Biofungicides
Liquid cultivation in batch-stirred tank reactors is the standardmethod of
producing microbial products. However, biofungicidesproductionrepresents
new territory in the progression of industrial fermentation technology.The
biocontrol agents must not only be produced in high yield but also should have
high retention of cell viability with maintenance ofcrop compatibility and
bioefficacy duringseveral months of storage. Mediumoptimization must
consider not only for spore yield but also for spore fitness based on qualities
suchasdesiccation tolerance, stability asa dry preparation and biocontrol
efficacy. Nutritional and environmental conditionsduringculturegrowth and
sporulation, which promote the accumulations of appropriateendogenous
reserves, may be a critical factor in determining spore fitness. Once harvested,
from culture, they must be stored, preferably in a dry,non-refrigerated state until
time for field application. Even after storage, the liquid culture-produced cells
must remain not only viable but active enough to rapidly colonize and establish
plant disease protection. The biocontrol application must be free of metabolites
that are phytotoxic and may have a detrimental effect on the crop (Slininger et
al., 1998).
Liquid fermentation is the preferred manufacturing process for
biocontrol agents. While fermentation of bacteria and yeast is straight forward
Biofungicides 191

and easily accomplished, this fermentation strategy presents difficult challenges


for fungi. In their natural environments, fungi grow along the surfaces or within
the structures of solid substrates and exhibit many different types of growth
phases and morphologies. When environmental conditionsare favorable and
sufficient amounts of nutrients are available, fungal growth occurs primarily by
the development of mycelia. However, when nutrients becomelimiting or
environmental conditions are harsh, the fungal mycelia stop growing and form
spores. Fungal growth in liquid media, particularly in fermentation vessels, is
vastly different from grownonsolidsubstrate.The mycelia have different
morphological properties and have a tendency to form clumps or pellets. The
occurrence of pellets greatly reduces the efficiency of fermentation and the final
spore yields. Although, the vegetative hngal mycelia can be used as an active
ingredient in product formulations, the mycelia can not easily be formulated or
stored for extended periods of time. Consequently, many efforts have been
placed on developing fermentation processes which maximize fungal spore
production (Stowell, 1991). Harman and coworkers of TGT Inc. (Geneva, NY)
successfully developed a large-scale fermentation technology combining liquid
fermentation with a secondary solid fermentation. It resulted in an inexpensive,
highly active biomass with a good shelf life. This method is now used in the
commercial production of biofungicide T. harzianum KRL-AG22(BioTrek
22G@) (Tronsmoand Hjeljord, 1998).

9 Formulation of Biofungicides
Intact-cell based biofungicides require very delicateandrather sophisticated
approachesin the design of commercial formulation. It is necessary that the
formulation ensure product stability during storage (i.e. shelf life), as well as
reliability during application. The formulation of biofungicideshasbeena
subject matter of two recent review articles (Lumsden et al., 1995; Fravel et al.,
1998).
Spores are natural survival vessels that can bereadilydried and re-
suspended in liquid delivery systems at the time of use, such as thoseof Bacillus
species. Some non-spore forming microorganisms such aspseudomonads, do
not possess the survival mechanisms of spores. Thus, drying and rehydration is
not possible with non-spore formers and offers formulation
challenges
especially with regard to stability. Therefore, lack of successful, viable
formulations of fluorescent pseudomonads remaina major obstaclefor their
large-scale use.
192 Chapter 7

9.1 Formulation of FungalAntagonists

Commercial preparations of the fungus Gliocladium virens (GlioGard@)


appeared in the market asan alginate prill formulation.Subsequently, this
formulationwasdiscontinueddue to certain qualitycontrolproblems.The
modified formulation (SoilGard@) produced by fluid-bed granulation included
dextrin as a binder and reduced content of alginate. It was reported to be stable
and highly effective product for controlling damping-off of various ornamental
andvegetable transplants causedby the pathogens Pythiumultimum and
Rhizoctoniasolani (Lumsden,et al., 1996).Alginategelhas also beenused
successfullytoprepareformulationsofbiocontrolbacteria as well as other
fungi, including pseudomonads, Trichoderma spp., G. virens etc. (Marois et al.,
1989).
Ecogenhas
adopted
proprietary
a formulation
technologyto
incorporate the yeast Candidaoleophila into awater dispersible granule
formulation(Aspire@) that provides stability duringstorage as well as user-
friendliness toaproduct.On the otherhand, Ampelomycesquisqualis, a
pycnidial parasite of powdery mildew fungi is formulated as a wettable powder
(AQ10@).Non-pathogenic Fusarium oxysporum is produced as a microgranular
product (Fusaclean@). Similarly, Pythium oligandrum has been formulated as a
granularorpowderproduct(Polygandron@)foruse as aseedtreatment on
sugarbeet for control of pathogenicPythium spp.

9.2 Formulation of BacterialAntagonists

Bacterial antagonists have been formulated in a variety of ways to control plant


pathogens. The sporulating gram-positive bacteria offer biological solutions to
the formulationproblems that haveplagued biocontrol. Kodiak'', awettable
powder formulation of Bacillus subtilis strain GB0 3, is highly effective for
control of the pathogens Fusarium and Rhizoctonia, as well as in stimulating
plantgrowth.Thegram-positivemicroorganism offers heat-anddesiccation-
resistant spores that can be formulated readily into stable dry powder products.
On the other hand, non-spore-forming organisms are moredifficult to formulate,
as they do nothave the survivalmechanismsofspores.Thegram-negative
microorganisms such as Pseudomonas syringae or Burkholderia cepacia have a
short shelf life and are readily killed by desiccation. These microorganisms are
traditionally formulated into various solid carriers. The different strains of P.
syringae are formulated as wettablepowderformulations(BioSave@ 10,
BioSave@l1) for post-harvest application tocitrus and pome fruit. Pseudomonas
fluorescens strain A 506 is supplied as apowder(BlightBan@ A 506) for
spraying onto leaf surfaces of apple and pear trees to provide protection from
Biofungicides 193

frost andfrom Erwinaamylovora, causeof fireblight. Mycostop@,based on


Streptomyces griseoviridis strain K 61 contains mycelium and spores andis also
formulated as a wettable powder. It controls damping off and root and basal rot
diseasesofornamentalsandvegetablescausedby Fusarium,Pythium and
Phomopsis.
Inviewof the user-friendlynatureof liquid formulations,newer
developments are being made in liquid media - either aqueous or mineral oil.
Theseformulationsallow for slow,continualgrowthoftheorganism in the
liquid or suspend growth to a starved level (Wacek, 1997). Examples include,
Pseudomonas fluorescens, formulated as an aqueous suspension of fermenter
biomass (Conquer@, Victus@), sprayed into mushrooms to prevent blotch caused
by Pseudomonastolassil. Similarly, B. cepacia is formulated asa liquid
suspension (Deny@)for seedling drench or use as drip irrigation for a few high-
value crops, suchas strawberry and melons.

10 Concluding Remarks
The promise of biologicals in large-scale agricultural market is beginning to be
realized, at least partially. It wouldbe unrealistic to expect that biological
control agents can completely replace chemical fungicides in disease control.
There are, however, areas in which biological control is superior to chemical
fungicide control. Presently, biofungicides occupy a niche market and provide
reasonable level of control as part of an integrated disease managementstrategy.
For biological control methodsto emerge as an economically viableoption, their
consistency and efficacy in controlling soilborne and foliar fungal diseases and
post-harvestdecay,needstobeenhancedtoa level comparableto that of
synthetic fungicides.Moreknowledge is needed to understandthecomplex
modesofactionof the antagonistic strains andtoapplythem in the best
conditions to achieve optimal biological control of fungal diseasesin the plants.
One way to improve efficacy and consistency of biological control would be to
mimic the complexity of the mechanisms operating in suppressive soils, and to
use several populations of antagonistic microorganism together. Improvement of
field performance of biofungicides to display curative or eradictive activity is
also necessary, to find commercial usage. For this, a strong collaboration and
understanding between the agriculture, industry and industrial microbiologists
are required to continue the advances of newbiologicals,

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47. Stowell, L. J., 1991.Submergedfermentation of biologicalherbicides, in
Microbial Control of Weeds, D. 0. TeBeest, ed., Chapman & Hall, N. y . , pp.
225-26 I .
48. Thomashow, L. S. and D. M. Wellcr,
1990. Role
of
antibiotics and
siderophores in biological control of take-all disease of wheat,Plaut Soil, 129,
93-99.
49. Tronsmo, A. and L. G. Hjeljord, 1998. Biological control with Trichoderma
species, in Plant-Microbe Interactiorzs and Biological Control, G. S. Boland
and L. D. Kuykendall, eds., Marcel Dekker, New York, pp. 1 1 1-124.
50. Tronsmo, A. 1989. Trichoderma lrarzra~~zc~~z usedforbiologicalcontrol of
storage rot on carrots, Norwegian J. Agric. Sci., 3, 157-161.
51. Utkhede, R. S., 1996. Potential
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problems of
developingbacterial
biocontrol agents, Can. J. Plant Pathol., 18,455-462.
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& Hall, pp. 255 - 256.
54. Verhaar, M. A,, P. A. C. van Strien and T. Hijwegen, 1993. Biological control
of cucumber powdery mildew (Sphaerotheca jiuligirrea)by Verticillirtrlr lecanii
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16(1 I), 79.
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R. Goss, M. J. Hopkinson andH.M. Collins, eds., AmericanSocietyfor
Testing and Materials,pp. 94-97.
56. Weller, D.M., 1988. Biological control of soil-borne plant pathogens in the
rhizosphere with bacteria, Annu. Rev. Phytopathol., 26,379-407.
57. Whipps, J. M.,1997.Developments in thebiologicalcontrol of soil-borne
plant pathogens, in Advances in Botarzical Research, Volunre 26, J. A. Callow,
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59. Wilson, C. L., 1997. Biological control and plant diseases - a new paradigm,
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60. Wilson, M.,1997. Biocontrol of aerialplantdiseases in agricultureand
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156.
This Page Intentionally Left Blank
Bioherbicides

1 Introduction
The use of herbicides with documented adverse affects suchasresiduesin
ground water and persistence of some herbicides beyonda single growing
season affecting rotational crops have led to increased interest in alternatives
suchas biological weed control. Biological herbicides offer yet another
beneficial trait, i.e. a killing action that is different from synthetic herbicides, so
they shouldbe efficient tools in combating weed resistance to specific
chemicals.
Hoagland (1996) defined bioherbicides as plant pathogens,phyto-
toxins derived from pathogens or other microorganisms appliedtocontrol
weeds. The whole foundation of biological weed control is to push the disease
process by tipping the ecological balance in favor of the pathogen. This is often
done to identify the right pathogen or the one that will work in diverse
environments.
A pathogen could qualify as the active ingredient for acommercial
product only if it canbeapplied in a viable form, atinoculum levels high
enough to initiate an infection and by manipulating the micro-environment for a
long enough period of the time. It is required to make sure that an infection gets
to the point that it can perpetuate itself. This would involve developing
formulations that allow them to work consistently under diverse field conditions,

199
200 Chapter 8

including stability of living organisms that would survive a distribution process


for as long as 18months between packaging and use.
The majority of bioherbicide candidates studied
are the fungal
pathogens. Nevertheless, recently it has been shown that itis possible to
consider the use of bacteria also as bioherbicide. The future potential of
mycoherbicides, in particular, is seen in areas that are served inadequately by
chemical herbicides. These areas include (1) control of parasitic weeds; (2)
control of weeds closely related to crops (crop mimics), in which case a high
degree of selectivity is necessary; (3) control of weeds resistant to chemical
herbicides and (4) control of weeds infecting small, specialised areas where
development of chemical herbicides would be too costly(Templeton et al.,
1986).
Several reviews on bioherbicides have appeared in the literature
recently (Mortensen, 1998; Hoagland, 1996; Christy et al., 1993, Gardner and
McCoy, 1992 and Zorner et al., 1992). In this chapter, an overview on various
bioherbicides that have been registered and commercialised is provided.

2 Fungal Bioherbicides
Fungi are capable of entering the plant host through wounds, through openings
in the epidermis (for example, stomata, nectaries), or by direct penetration of the
cuticle by germinating spores. Development of phytopathogenicfungias
mycoherbicides has progressed well. Charudattan (1991) has cited identification
and development of over 109 pathogens, out ofwhich 73 organisnls having been
placed in commercial development programs in thedecade of 80s. Self-
disseminating pathogens (such as rusts, smuts) as well as poorly disseminating
pathogens, such as soil-borne and mucoid-spored fungi, have been explored.
Bioherbicidal pathogens being studied presently are host-specific for
reasons of environmental safety. Extreme host-specificity is an advantage where
a weed is closely related to the crop, in which it is to be controlled. However,
there may not be enough perspective host-specific pathogens for each problem
weed. Besides, use of host specific agents will be precluded for economic reason
alone due to presence of multiple weed species with each crop. Therefore, only
broad-host range pathogens are likely to compete well with broad-spectrum
herbicides, by virtue of their targeting multiple weedspeciesinsingle
application (Sands and Miller, 1992). The well-known broad-host range plant
pathogens include, Sclerotinia sclerotiorum,
Pythiumultimum.
Sclerotium
rolfsii,Phymatotrichumomnivorunz and Pseudomonassolanacearum. Often
soil-borne, these pathogens are capable of surviving in the soil after killing one
plant until they attack another susceptible plant of the same or different species.
Their mode of action involves root or crown invasion, vascular plugging and
Bioherbicides 201

wilting. However, as with other plant pathogens, the mechanisms involved in


host range restriction, are unknown.
Daniel et al. (1973) noted that fbngi selected
candidate
as
mycoherbicide should possess certain essential characteristics. The fbngus must
beamenableto invitro production. The product must alsoremainstable in
culture as well as in storage, have no dormancy factors that limit infectivity, and
be able to infect weed hosts in a relatively broad spectrum of environments.
Fungipossessingthesecharacteristicsaregenerallyfacultativepathogens.
However, only a few of these organisms have been commercialised (Table8.1).
A . cassiae is effective against sicklepod (Cassia obtusifolia) in soybean
grown in diversity of soil types and environmental conditions. It causes a foliar
blight disease in host plants and is being developed by Mycogen Corporation
under the trade name Casst@ for sicklepod control.
Collego@ was developedin a cooperative effort between the University
of Arkansas,USDAand private industry. It ismarketedas a complex
formulation of dried conidia of C. gloeosporioides f. sp. Aeschynomene.

Table 8.1 Registeredfungalbioherbicides

Trade
Target Pathogen

Alternaria
cassiae Sicklepod
Soybean Casst Mycogen Corp.
(Cassia San Diego, CA
obtusrjblia)

Colletotrichunl Northern Rice and Collego Pharmacia &


gloeosporioides f. jointvetch irrigated Upjohn
sp. aeschynotnene (Aeschynomene soybean Kalamazoo, MI;
virginica) Encore
Technologies,
Minnetonka, MN
Phytophthora Stragler vine Citrus DeVine Abbott Labs.
palmivora (Burl.) (Morrenla groves Chicago, IL, USA
odorata)
Colletotrichunl Round-leaved Vegetable BioMal Philom Bios
Gloeosporioides mallow (Malva crops and Saskatoon, Canada
f. sp. tnalvae pusilla) straw-
berries
Puccinia Yellow Sugarcane, Dr. Tifton Innovation
Canaliculata nutsedge maize, BioSedge Corp., Tifton, GA
(Schw.) (Cyperus potato,
esculentus L.) cotton and
soybean
202 Chapter 8

C. gloeosporioides is a facultative sporophyte that causesa lethal stemand


foliage blight of its host weed when inoculated with spores. The formulation is
rehydrated in aquoussolutions. It is applied aerially to the surface of rice
paddies, when s t e m of northern jointvetch begin to exceed the height of the
rice, prior to initiation of the flowering of the weed. Within 2 weeks, Lesions
formon the stems of the weed, followed rapidly by weed death. College@
provides long-term control of northern jointvetch, curbing the need for periodic
reapplication.
Similarly, Devine@, the commercial bioherbicide based on
Phtophthora palmivora is formulated as a fresh preparation of the biocontrol
agent by Abbott Laboratories. P. palmivora is a facultative parasite that
producesa lethal root rot of its host plant (strangler vines) andpersists
saprophytically in the soils for extended period of time. The fungus was first
isolated from milkweed vine (Morrenia odorata) in Florida and is registered
exclusively for its control in Florida citrus. Devine@is a liquid preparation of
viable chlamydospores produced in submerged culture. It is applied as a post-
emergent herbicide to the soil around citrus trees to infect seedling and mature
vines within 2-10 weeks after application. The infection process begins at the
soil line and moves down to attack the roots, eventually overpowering even the
largest strangler vines without damaging the citrus trees. Control is usually
100% and could last for over 2 years (Charudattan, 1988).
BioMal@,a fungal bioherbicide based aon Colletotrichum
gloeosporioides f. sp. malvae was developed by Philom Bios, aSaskatoon
biotechnology company. It infects only round-leaved mallow (Makva pusilka)
which is a problem weed in Western Canada and is very effective at killing it.
On application to the field, the fungal spores settle on the leaves of the plant and
germinate growing mycelium. These thread like extensions act as little hooks,
securing themselves to the outside of the leaf. From there, the fungus penetrates
the leaf and infects the entire plant. The weed rots and is killed due to inability
of taking up water or nutrients. The fungus eventually grows more spores that
are carried by the wind to find another Round-leaved Mallow plant to infect. C.
gloeosporioides breaks through the leaf structure of round-leaved mallow but
cannot invade other plants. This is important as round-leaved mallowoften
grows in agricultural crops that are closely related. In this situation, the use of
chemical herbicides is limited because the agricultural crops may be damaged.
Applying BioMal eliminates the weeds without riskto the crop or the
environment.
Nutsedges (Cyperus spp.) comprise agroup of commonlyoccurring
weeds that are among the most difficult to control. Chemical weed control
programs are seriously inadequate to control nutsedges. Frequently, the weed
germinates below the treated zone and avoids herbicide injury. An alternative is
offered by the use of microbes that have herbicide activity specific for the
problem weeds and do not infect desirable plants. Yellow nutsedge (Cyperus
Bioherbicides 203

exculentus) is a serious or principal weed of sugarcane, maize, potato, cotton


and soybean, chiefly in southern Africa and North America. Dr. BioSedge, a
bioherbicide based on Puccinia Cannliculata (Schw.) has been reported to be
effective in controlling yellow nutsedge. It has been commercialised by Tifton
Innovation Corporation(Tifton,GA). Kadir and Charudattan (1999) have
obtained a patent on the application of a fungal pathogen Dactylaria higginsii
(Luttrell) M. B. ellis which is claimed to control Cyperus spp. including, purple
nutsedge (C. rotundus), yellow nutsedge (C.esculentus), annual sedge, globe
sedge and rice flat sedge (C. iria). Purple nutsedge is particularly known to be
an aggressive weed that is resistant to control measures.
Scheepens et al. (1996) of Ciba-Geigy have obtained a patent of fungal
pathogen Ascochyta caulinn acting as mycoherbicide for control of weeds of
familly Chenopodiaceae in crops, especially annual herbaceous weed
Chenopodium album.
These fungal bioherbicides require specific conditions for infection and
disease development, such as a prolonged dew period in excess of 24 hours.
Synergistic interactions of chemicals, mostly herbicides and plant growth
regulators, and fungal weed pathogens have been discussed in Chapter 10. Such
interactions could help provide improved bioherbicidal weed control and reduce
the amount of herbicide application. For example, Alternaria cassine functions
as a fungal bioherbicide to control sicklepod, and shows enhanced control in
presence of glyphosate. Glyphosate suppresses the defence response of the weed
by lowering phytoalexin production and thus acts synergistically with the
pathogen.

3 Bacterial Bioherbicides
Bacterial bioherbicides research is focused primarily on use of a rhizobacteria.
In contrast to fungal bioherbicides using foliar plant pathogens, which have been
mostly directed towards dicotyledonous weeds, rhizobacteria have been directed
towards grassy weeds (monocotyledons) in cereal crops. Rhizosphere strain of
Pseudomonas flourescens inhibited germination and growth of downy brome
(Brontus tectorum) without affecting the growth of wheat. Kremer et al. (1990)
demonstrated rhizobacteria representing diverse gram-negative bacterial genera,
as potential biocontrol agents for broad leaf weeds.
Zorner et al. (1996) of Mycogen Corporation reported development of
two gram-negative bacteria, namely Pseudomonas syringue pv. togetes (PST)
and Xanthomonas compestris pv. poaea (X-PO). PST demonstrated an ability to
control a variety of weeds in the family Asperogens, while X-PO has been found
to be specific to annual bluegrass (Poa nnnua L.). Neither of these organisms is
dependent on a dew period for host colonisation and they both demonstrate their
efficacy under field conditions. Xanthornonas campestris, a vascular
204 Chapter 8

phytopathogenic bacterium is applied in the early spring to newly mown grass.


The bacterium rapidly colonises the xylem, the foliage wilts and the plant die
within six weeks. Theorganismrequires
prolonged
a period
of
warm
temperature and some form of mechanical wounding to allow penetration for
disease progression. The strain controls blue grass in turf but does not harm
desirable turf species.
Christy et. al. (1993) reported development of combinationsbacterial
of
and chemical agents for enhanced weedcontrol. This approach has been termed
asthe X-tendbioherbicidesystembyCropGenetics International, a U.S.
biotechnologycompanydeveloping it. A rangeofbacteriaandherbicide
combinations has
been tested observing synergistic interactions in both
greenhouse and field trials (see Chapter 10, Section 3.3). Nevertheless, a need
for more robust strains or improved formulationsis felt for commercialisation.

4 Environmental Limitations for Efficacy


of Bioherbicides
Both, fungal bioherbicides, as well as bacteria based bioherbicides, suffer from
environmental limitations for efficacy under field conditions. A fungus requires
specific conditions for infection and disease development. Some period of dew
on the surfaceof the plant is often required, so that the fungalsporecan
germinate and infect the plant. The dew periods of known fungal bioherbicides
range from 8 to 72 hours. On the other hand, it may be possible to escape the
limitation of a dew requirementin case of bacterial bioherbicides. The infection
processoccursthroughmovementof the bacteria into the hostviaa direct
woundintroducedbyamowingoperationorsomeform of mechanical
wounding to allowpenetration inthe host for colonisation. However,the
bacteria also requireaprolongedperiodofwarmtemperatureand this may
prove to be a limitation in cooler climates (Figure 8.1). Since all bioherbicides
are living organisms, it is likely that all potential pathogenic agents will carry
some sort of environmental limitation on efficacy, a fact that needs to be dealt
with effectively.
Prolonged Fungal spore
Fungal periods dew fa
germination and
bioherbicide
(8 - 72plant
infection
hrs)
of

Prolonged period of Mechanical


Bacterial to penebak
bioherbicide requires r, warm
ternpetature + wounding of
(-50-60days) target

Figure 8.1 Environmental limitations of bioherbicides


Bioherbicides 205

5 Production of Bioherbicides
Product variability during production can be one of the reasons for unreliable
field performanceofamicrobial herbicide. An effective biologicalcontrol
productwould be consistentlyproducedandhaveashelf life of 1-2 years.
Production and formulation are closely inter-related in the development of cost-
effective inoculants. Inoculant is usually mass-produced by submerged
fermentation. However, as spores are preferred for formulation, thls method is
used typically for those fungi that sporulate in the submerged state.
Temperature,aerationand the balancebetween nutritional elementscontrol
sporulation of filamentous fungi in submerged culture. Controlled culture in the
homogenous medium in bioreactor
a is monitoredand
manipulated
biochemically to improve efficacy of inoculant.
Mostcommonly,inoculants are spores, separatedfrommediumand
mycelium by filtration and centrifugation. In Collego', 8 0 4 5 % of the
propagulesare fission spores, 8-10% areconidiaand 5% blastosporesplus
arthrospores (Churchill, 1982).Mycelialinoculantscan be effective, as with
DeVine', butlackofshelf life precludes its widespreaduse. Alternatively,
mycelium can beharvestedand treated to induce sporulation, but this adds
significantly to the cost.
The target shelf life of 1-2 years has been achieved by drying spores
that have been harvested from submerged culture and mixed with inert filler
such as kaolin. However, difficulties have been encountered in stabilising some
fungal spores, including those of Colletotrichum tuncatum (Jackson et al., 1996)
and Phytophthora palmivora(Kenney, 1986).
Some microbial herbicides have been produced on materials such as
wheat, straw, oat grains and corn meal. However, propagules in solid substrate
may be difficult toseparateor extract for formulation. Alternatively, the
substrate may become part of the final product, as with some insect pathogenic
fungi. Nevertheless, solid substrate fermentationmaybe the onlymethodto
produce some fungi.

6 Formulation of FungalBioherbicides
Development of reliable and efficacious fungal bioherbicidal fommlation can
oftenbechallenging.Oneof the goals is tokeep the propagulesviableand
inactive for reasonable
a lengthof time, say 1-2 years. To date, most
bioherbicide formulations are concentratedon maintaining fungal agent viability
in storage and reducing dew requirements. DeVine' is an aqueous concentrate
of chlamydospores of Phytophthora palmivora. It is being used for control of
stanglervine (Morrenia odorata) in citrus orchards in Florida. The product is not
206 Chapter 8

very stable and has a shelf life of only 6 weeks, when the product is refrigerated.
Collego@is a two component formulation. One package component consists of
dried spores of Colletotrichum gloeosporioides f s p . aeschynomene in a wettable
powder. A second package contains rehydrating medium. The product is
reconstituted in a two-step process by first preparing the rehydrating solution
then adding the dryspores and mixing well. BioMal@ is a wettable powder
composed of dried spores of Colletotrichum gloeosporioides in silica gel added
directly to spray tank. Cas@ is formulated as spores of Alternaria cassiae in
emulsifiable paraffinic oil. Conidia of some fungi have proven to be difficult to
stabilize in dry formulations and for these, more resistant formulations have
been chosen. Among these, oil-based suspension emulsions and invert
emulsions have attracted most attention.

6.1 In vert Emulsion Form ulation

The biocontrol efficacy and practical usage of most fungal bioherbicides are
adversely affected by lengthy dew requirements, which range from 8 to 72 hours
or more. The elimination or reduction of dew requirements should improve the
biological control potential of fungal bioherbicides. One approach that has
shown promse in reducing dew requirements of bioherbicides, involves the use
of invert emulsion formulations. An invert emulsion consists of water suspended
in oil, in contrast to a standard emulsion,in which oil is suspended in water. The
ability of inverts to trap water around fungal spores and retard evaporation of
water spray droplets during and after application could prove beneficial for their
use with bioherbicides.
Boyette et al. (1993) have studied the efficacy of an invert formulation
for the pathogen Colletotrichum truncatum for weed control of Hemp sesbania
(Sesbaniaexaltata), a perennial weed of cottonandsoybean under field
conditions. Theoil phase of the invert emulsionconsisted of parafinicoil,
monoglyceride emulsifier, parafinic wax andlanolin.Theaqueousphase
contained conidia suspended in distilled water. The two phases were mixed at a
ratio of 2:3 by volume, aqueous phase:oil phase. They reported an optimal weed
control that was as good as the level of control achieved with acifluorfen, a very
effective chemical herbicide (Figure 8.2) (Table 8.2).
Amsellemet al. (1990) reported similar enhancedefficacythrough
invert emulsionformulation of fungal bioherbicide Alternariacassiae for
controlling sicklepod. However, it was found that the non-pathogenic fungi were
able to infect a variety of plants, when applied in the invert emulsion. They
concluded that specificity of A. cassiae was abolished either due to physical
damage to the host plant cuticle, or suppression of the host defence responses,
and that an invert emulsion of A. cassiae in the field could cause non target
damage.
Bioherbicides 207

Invert emulslon retard Evaporation of reducin or


dew
of fungal water
____c spray eliminahg requirement
bioherbicides

Invert emulson of con@olled Hemp =sbanla rable Acifluorfen


c. tmncatum w
-. (Sesbania exaltata) to t (an effective
(fungal bioherbiade) a perennial weed herbiade)

Figure 8.2 Invert


emulsion
formulationof
fungal
bioherbicides
display
improved biological control potential

Connick et al. (1991) reported development of an invert emulsion that


exhibited
lower viscosities and greater water retention properties.
Also,
vegetable oils could beused to enhance efficacy of fungal bioherbicides, such as
Colletotrichum orbiculare, for control of spinycocklebur(Auld,1993).This
invert emulsiondemonstrated improved spreading properties and no
phytotoxicity.

6.2 Oil-basedSuspensionEnlulsions

Oilsuspensionemulsions of bioherbicides have beeninvestigatedas less


expensive, easy to prepare alternatives to oil invert emulsion formulations, that
couldbeapplied with conventionalsprayequipmentandeffectivelyused at
relatively reduced volumes. Similar formulations have been very effective for
Btk at low volume application and gave lower rate of evaporation. Egley and
Boyette (1995), however found that suspension emulsions were inferior toinvert
emulsions as a water source for bioherbicide activity, in the absence of a dew
period.

Table 8.2 ControlofHempsesbaniawith C. truncatum and Soybean


yield under field conditions

rate
Carrier sesbania
Hemp Soybean
Spray freatnlerzt
(l/ha) (Ayield
mortality) (kg/lta)

94 19 1400
Conidia / water
I87 40 2160
94 72 2181
Conidia / Invert
187 97 2593
Acifluorfen 2618 187 98
208 Chapter 8

7 Formulation of Bacterial Bioherbicides


Oneof the challengesconfronting theuse ofplantpathogenicbacteria for
biological weed control is the requirement of free water for dispersal and the
need for wounds or natural openings, such as stomata, hydathodes or lenticels,
for entry of the bacteria into the plant (Johnson et al., 1996). The majority of
plant pathogenic bacteria are more environmentally sensitive than most fimgi,
which often have pigmented spores or structures that are naturally adapted to
withstanding environmental stress, such as UV radiation and desiccation.
Zidack et al. (1992) reported that organosilicon surfactant Silwet L-77
facilitated the penetration and entry of bacteria directly into the weed (without
wounding) via open plant stomata and hydathodes. A low surface tension, 30
dyneslcm or loweris required for penetration of liquid into the stomata of a leaf.
Silwetreduces the watersurfacetension to 20 dyneskm. Formulationof
Pseudomonas syringae pv. Tagetis (PST)with this surfactant resulted in
significant increases in disease severity and incidence in perennial weed such as
Canada thistle (Cirsium awense), when compared to the bacterial formulation
without the surfactant. It has been suggested that delivery of bacteria into these
natural openings protect them from UV irradiation and desiccation.

8 Concluding Remarks

Thecommercializationofmycoherbicidessuch as Devine@and Collego@


illustrates the potential of the use of phytopathogenic agents as bioherbicides to
control selected weeds. However, bioherbicides are viewed as complimentary
adjuvants to current weed management practices rather than as alternatives to
chemical herbicides. It has been suggestedthat development of contained broad-
host range pathogens might better compete with broad spectrum herbicides by
virtue of their useagainstmultiple target weeds,reducedresidue risk and
possibly perceivedaspects of safety (Sand and Miller, 1993).
Further research on formulation of fungal bioherbicides to overcome or
reduce dew requirements is also necessary. A need for shorter dew periods can
improve reliability and efficacy, as well as reduce inoculant dose requirement
(Amsellem et al., 1990).

References

I. Amsellem, Z., A. Sharon, J. Gresseland P. C. Quimby, Jr., 1990. Complete


abolition of hlgh inoculum threshold of two mycoherbicides (Alternaria
cassiae and A . crassa) when applied in invert emulsion, Phytopat/~dogy,80,
925-929.
Bioherbicides 209

2. Auld, B. A,, 1993. Vegetable oil suspensionemulsions


reduce
dew
dependence of a mycoherbicide,Crop Protect., 12,477-479
3. Boyette,C. D.,P. C.Quimby,Jr.,C.T.Bryson,G. H. Egleyand F. E.
Fulgham, 1993. Biological control of hemp sesbania(Sesbania exaltata) under
field conditions with Colletotrichunr truncatunr formulated in an Invert
emulsion, Weed Sci., 41,497-500.
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formulation
and
spraying
systems
improvement
for of
mycoherbicide, Weed Technol., 10(3), 637-644.
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invert emulsion withhighwater retention for mycoherbicide delivery, Weed
Technol., 5,442-444.
11. Daniel, J. T., G. E. Templeton, R. J. Smith and W. T. Fox, 1973. Weed Sci., 21,
303-307.
12. Egley, G. H. andC. D. Boyette,1995.Water-cornoilemulsionenhances
conidia germination and mycohcrbicidal activity of Collefotrichztnt tntncatunr,
Weed Sci., 43, 312-3 17.
13. Green, S., S. M. Stewart-Wade, G.J. Boland, M.P. Teshler and S.H. Liu, 1997.
Formulating microorganisms for biolological control of weeds, in Platrt-
14. MicrobebtteracfiortsandBiological Control, G. J. Bolandand L. D.Kuy
Kendall, eds., Marcel Dekker, Inc., New York, N.Y., USA, pp. 249-281.
15. Gardner, W. A.and C. W. McCoy,1992.Insecticidesandherbicides, in
Biotechnology of Filanrentous Fungi;
Technology and Products, D. B.
Finkelstein and C. Ball, eds., Butterworth - Heinemann, Stoneham, MA, USA,
pp. 335-359.
16. Hoagland, R. E. 1996.ChemicalInteractions with bioherbicides to improve
efficacy, Weed Technol., 10, 65 1-674.
17. Jackson,M.A,, D.A. Schisler, P. J. Slininger et al.,1996.Fermentation
strategies for improvingthefitness of a bioherbicide, Weed Tech., I O , 645-
650.
210 Chapter 8

18. Johnson, D. R., D. L. Wyseand K. J. Jones,1996.Controllingweedswith


phytopathogenic bacteria, Weed Technol., IO, 62 1-624
19. Kadir, J. and R. Charudattan,1999.Control of Cyperus spp.withafungal
pathogen, U.S. Patent 5,945,378 (August 31, 1999).
20. Kremer, R. J., M. F. T. Begonia, L. Stanley, E. T. Lanham, 1990.
Characteristicsofrhizobacteriaassociatedwithweedseedlings, Appl.
Environ. Microbiol. 56, 1649-1655.
21 Pilgeram, A. L. and D. C. sands,1998.Mycoherbicides, in Biopesticides:
Use and delivery, F. R. Hall and J. J. Menn, eds., Humana Press, Totowa,N. J.,
pp. 359-370.
22. Mortensen, K., 1998.Biologicalcontrolofweedsusingmicroorganisms, in
Plant Microbe Interations and Biologrcal Control,G. J. Boland and L. D. Kuy
Kendall, eds., Marcel Dekker,New York, pp. 223-248.
23. Sands, D. C., and R. V. Miller, 1993.Altering
the
host
range
of
mycoherbicides by geneticmanipulation, in PestControl with Enhanced
Environnwftal Safety, S. 0. Duke,J. J. Mennand J. R. Plimmer,eds.,
American Chemical Society, Washington, D.C. pp. 101-109.
24. Templeton, G. E., R. J. Smith Jr., and D. 0. TeBeest,1986.Progressand
potential ofweed control with mycoherbicides, Rev. WeedSci.,2, 1-14.
25. Zidack, N. K., P. A. Backman and J. J. Shaw, 1992. Promotion of bacterial
infection of leaves by an organosilicon surfactant: Implications for biological
weed control, Biol. Control, 2, 1 1 1 - 1 17.
25. Zorner, P. S., S. L. Evans and S. D. Savage, 1992. Perspectives on providing
a realistic technical foundation for the commercialisation of bio-herbicides, in
Pest Control with Enhanced Environr~~ental Safety, S. 0. Duke, J. J. Menn and
J. R. Plimmer, eds., American Chemical Society, Washington, D.C., pp.79-86.
26. Zorner, P. S., S. D.Savage, S. L. Evansand P. Simpson, 1996. Bacteria as
biological herbicide, Abstracts, SOC.Industr. Microbiol. Meeting, S. 10.
Mycoinsecticides

1 Introduction
Many insect pests aresusceptible to infection by naturally occurring insect
pathogenic fungi. Several fungi have been studiedas potential mycoinsecticides.
These h n g i are very specific to insects, often to particular species, and do not
infect animals or plants. Fungi provide a needed control of insects with sucking
mouth parts. While, bacteria and viruses mustbe ingested to cause disease, fungi
can cause infection by penetrating the outer structure of insects. Fungi provide
the only satisfactory microbial means of biocontrolof plant sucking insects such
as aphids and white flies that are not susceptible to bacteria and viruses. These
are living, infectious microbialagents that havecontact activity like many
chemical insecticides. However, they are slow acting and take about 3-7 days to
kill their insect hosts; in this regard their use is analogous to insect growth
regulators.

1.1 Mycoinsecticides for Agricultural and Forest insect Pests

McCoy et al. (1988) listed high virulence, broad host range, amenabilityto mass
production and formulation, storage and product stability as factors that fungi

21 1
212 Chapter 9

mustpossess to bepotentialmycoinsecticides.Several fimgi havebeen


discovered that possess these attributes and
have
been
developed
asmycoinsecticides. The well-studied insect pathogenic fungi include Beauveria
bassiana for white flies, locusts and beetles, Metarhiziumanisopliae and M.
Javoviride forlocustsand Verticilliumlecanii forcontrol of aphids.Other
possible fungal candidatesinclude B.
brongniartii,
Hirsutella
thompsonii,
Paecilomycesfumosoroseus.P.farinosus,NomuraearileyiandAschersonia
aleyrodis.
Deuteromycetes (imperfect fungi) such as Metarhizium anisopliae and
Beauveriabassiana are the causativeagents of green and whitemuscardine
diseases,respectively. B.bassiana is acommonsoil-bornesaprophytefungus
that occursworldwide. It attacks a wide range ofbothimmatureandadult
insects. The extensive list of hosts includes such important pests as white flies,
aphids,grasshoppers, termites, Coloradopotato beetle, Mexicanbeanbeetle,
boll weevil, cereal leaf beetle, bark beetles, lugus bugs, chinch bug, fire ants,
European corn borer, codling moth and Douglas fir tussock moth. There are
manydifferentstrains of the fungus that exhibitconsiderablevariationin
virulence, pathogenicity and host range.
Metarhizium spp. is a natural enemy of corn root worm, white grubs
and some root weevils. It also has a very broad host range and is extensively
used in Brazil against spittle bugs in sugar cane and alfalfa. There are several
otherspeciesofinsect-pathogenicfungithat have beentestedasmicrobial
insecticides. Verticillium lecanii is used in Europe against greenhouse whitefly
and thripsandaphids,especiallyingreenhousecrops. Hirsutellathompsonii
infects mites and Entomophthora muscae infects flies.
Entomophagamaimaiga, a native ofJapan,is an importantfungal
pathogen of gypsymoth (Lymantriadispar) larvae. Underseveredisease
situations, this fungus can reduce gypsy moth populations as much as85%. The
fungus survives both the winterand the absence of suitable hosts as a thick-
walled 'resting spore' in the soil and on tree bark. The resting spores have been
used effectively to spread the fungus to gypsy moth infested sites. In summer,
resting spores germinate and produce sticky spores at the end of a stalk that
grows just above the soil surface. Gypsy moth caterpillars come into contact
with these sporesas they searchforsuitableleaves to feedon. The fungus
digests its way through the exoskeleton of the caterpillar and grows inside its
body. Infected caterpillars may die within one week. When young caterpillars
are affected early in the summer, the funguswill produce a second type of spore
called conidia. These microscopic spores are spread by the wind and can infect
other caterpillars. The cycle of conidia production and infection may occur four
to nine times during the summer (Hajek et al.,1996). Like most fungi, its spores
needmoisture and highhumidity to germinate.Temperatures of 50-80" F
enhance fungal growth.
Mycoinsecticides 213

1.2 Mycoinsecticides for Mosquito Control

Fungi such as Culicinornyces clavosporus, Tolypocladium cylindrosporum and


Lagenidium giganteum are pathogens of mosquito larvae (Gardner and McCoy,
1992). L. giganteum consist of asexual zoospores that are infectious to mosquito
larvae. Application of C. clavosporus conidia or hyphae to mosquito-infested
natural and artificial ponds in the United States and Australia has yielded 86-
100% control of Culex spp., Aedes spp., and Anopheles spp. The fungus can be
mass-produced as conidia or mycelia in surface or submerged culture and can
recycle in the mosquito population. Similarly, both conidia and blastospores of
T. cylindrosporum are infective tomosquitolarvaeand it has potential as a
mosquito mycoinsecticide.

2 Mode of Action
Insect pathogenic fungi have contact activity, much like chemical insecticides.
Their ability to invade actively the external skeleton or cuticle of insects, makes
them pathogen of choice for sucking pests, whose mouth parts may preclude
uptake of other pathogens such as bacteria and viruses (Figure 9.1). The site of
invasion by fungi is often between the mouth parts at inter-segmental folds or
through spiracles, where locally high humidity promotes germination and the
cuticle is soft andmore easily penetrated(Charnleyet al., 1997).Thehost
insects mostcommonlyaffected include, aphids, white flies, locusts and
grasshoppers, lepidopterous larvae, ants, termites and ground beetles in foliar
and soil habitats (Burges, 1998).
M. anisopliae and B. bassiatla have hydrophobic spores which appear
to bind to insect cuticle, as it is picked up by the insect from the environment
(soil or plant surface) during feeding or movement. Onceon the cuticle, the

Mycoinsectiades di la Contact
activity fm Sucking pests (such
(e.g. B. bassiana chemical
like conkol t as aphids,
thrips
and M. anisopliae) flies) whiteinsecticides
and

Insect body
1
incapable of
ingesting

Pathogens such
as bacteria
and
vimses

Figure 9.1 Contact activityof mycoinsecticides.


214 Chapter 9

Figure 9.2 Scanningelectron micrograph of Metarhiziumanisopliae


fungal sporesgerminating on the surface of cuticle of the
tobacco budworm (St Leger et al., 1988)).

spore responds to biochemical cues present (chemotaxis) in the waxy epicuticle


and germinates within 8-16 hrs. (Figure 9.2). Soon the fungus stops growing
horizontallyonthesurfaceofthecuticleandinitiatepenetration,usinga
combination of mechanical pressure and a mixture of cuticle degrading enzymes
(lipases, proteases and chitinases), which attack and dissolve the cuticle. Once
the fungus breaks through the cuticle and underlying epidermals, it tends to
invadeinhaemocoel(bodycavity)oftheinsectandproliferateinthe
haemolymph. The insect's
defensesystem in the
homocoel
employs
phagocytosis and the secretionof antagonistic compounds namely quinones and
melamines. However, once inside the insect, entomopathogenic duteromycetes
such as Beauverea produces a toxin called Beauvericin that weakens the hosts
immunesystem.Usually,within 24 hrs. ofgermination,thefungusrapidly
proliferates through theinsect. Growth can bein the form of mycelium or yeast-
llke blastospores. The infected insects stop feeding and become lethargic. They
may die relatively rapidly within 2-7 days (Figure9.3) (Jaronski, 1997).
The life cycle of the fungus is completed when it sporulates on the
cadaver of thehost.Undertherightconditions,particularlyhigherrelative
humidity,thefungus will breakoutthroughthebodywall oftheinsect
producing aerial spores. High humidity is critical to spore germination, fungal
survivorship and transmission from host to host. The dead insect's body may be
firm and "cheese-like" or an empty shell, often but not always with green, red or
Mycoinsecticides 215

- - -
Attachment
to cuticle

Chemotoxis of
Adhesion Germination

- -
growing
hyphal __c Generation -m Penetration
of enzymes
tip on cutide
Feeding
Proliferation stopage __c Death

Figure 9.3 Mode of action of the insect pathogenic fungus.

brown fungal growth, either enveloping the body or emerging from joints and
bodysegments.Theseexternalhyphaeproduceconidia that ripenandare
released into the environment completing the cycle. This may allow horizontal
or vertical transmission ofthe disease withinthe insect population.
Deathof an insect fromfungal infection is probably the result of
starvation or physiologicallbiochemical disruption brought about by the fungus.
For example, B. bassiana kill their host by depleting the insect's energy reserves
(physiological starvation). It also causesadecline in fecundity in Colorado
beetles that survive infection. Similarly, M. jlavoviride causesreduction in
feedingand flight of desert locusts. Anotherpossiblemechanism that may
contribute to the demise of the insect is the occurrence of secondary metabolites
that are insecticidal. Forexample, M. anisopliae produceavarietyof toxic
metabolites that act as neurotoxins (e.g. the destruxins) or general metabolite
disruptors(the viridoxins). It is reported that some insects infectedwith
entomopathogenic fungi crownto the top of the plant to die (summit disease).

3 Production of Mycoinsecticides
Production of entomopathogenic fungi on solid substrates give rise to conidium
that are the natural form of spored Hyphomycetes dispersed by rain and/or air
currents. This form is both stable and convenient for dispersion. On the other
hand, submerged, liquid culture fermentation produces blastospores, a yeast-like
phase of vegetative growth, that are both infectious and germinate faster than
aerial conidium. The blastospores are environmentally sensitive particularly to
desiccation. Thismakes their shelf life limited to fewa monthsunder
refrigerated conditions.VariousDeuteromycetesfungispeciessuch as B.
bassiana, M. anisopliae, M. jlavoviride, P. furnosoroseus and P. farinosus are
216 Chapter 9

mass produced through submerged,liquid culture fermentation. Mass production


of several entomopathogenic fungi has been reviewed (Bartlett and Jeronski,
1988).
For solid substrate production, nutrient rich cereal grainsprovide
maximum surface area on which conidia can be produced. Rice is widely used,
but millet is also found good as nutrient substrate for harvest convenience. The
grain is brokentomaximumsurface area, soakedorboiled to achieve the
requisite moisture content and sterilized. Heat provides increased availability of
nutrients to the fungus. The fungal bio-mass builds upin the broth, on the cereal
nutrients. Addition of calcium carbonate or calcium sulfate can increase the pH
and prevent grains sticking to each other. The grains can be supplemented with
nutrients ornutrient-sockedporousmineralgranulescanreplace it. Mineral
granulessuch as pumice,exfoliatedvermiculiteand clay, canberetained as
granularformulationordisintegrated to facilitate spraying(Guillon,1997).
Heat-sealablespawn-growingmushroombagsallow efficient aeration during
incubation and grain drying under near 100% relative humidity. The conidia are
well preserved duringthe sporulation period until harvest. Harvesting is done by
extractionofdrysporesby an air stream in afluid-beddeviceforminga
virtually pure spore powder. Expensive drying and harvesting can be avoided by
marketing the growing bags as end-use containers, even before sporulation has
peaked, providing
extended
shelf life bytaking
advantageof
survival
characteristic of conidia (Burges, 1998).
MycotechCorporation has developedacommercial-scaleproduction
facility for aerialconidiaof B. bassiana (WraightandCarruthers,1999).
Blastosporesproduced in a liquid medium in conventionalfermenter,are
incorporated into a proprietary solid medium that is loaded into trays in large
chambers with forced aeration in a controlled environment. Prohse sporulation
is initiated throughout the substrate within a few days. Afterthe culture matures,
the spores are dried withinthe chamber at a controlledrate to approximately 5%
moisture content and then harvested directly from the chamber. The extracted
product is anearlypureconidialpowdercontaining 1.2-1.8 x10" conidia/g.
This production technology is adaptable to other fungal pathogens that can be
mass-produced on solid substrates, especially Metarhizium and Paecilomyces
SPP.
Cultureconditionscaninfluence the characteristics offungalspores
and can be manipulated to increase mycoinsecticidal efficiency. Blastospores of
B. bassiann fromnitrogenlimited culture, werefoundmore virulent (lower
lethal time, LTSo) towardsrice green leafhopper, than blastospores from carbon
limited cultures (Lane et al., 1991).
Mycoinsecticides 21 7

4 Formulation of Mycoinsecticides
The mycoinsecticides can be formulated in a manner similar to conventional
pesticides, i.e. as foliar sprays, soil drenches, granules and baits. Nevertheless,
several unique features of microbial pest control agents have to be considered in
the formulation. The spores of entomopathogenic fungi are the agents of fungal
disseminationand infection in nature that are sensitive to desiccation. These
fungal spores have to be keptalive in a dormant state for a reasonable time. Also
that underfavorableenvironmentalconditions,thesefungihaverecycling
potential after initial application.

4.1 Dormancy of Fungal


spores

Thereare three essential componentsofconidialgermination(a) nutritional


source,(b)oxygenand(c)water.Eliminationofanyonecomponentwould
preventgermination, yet killing the conidiumwould be avoided.Residual
nutrients in a typical mass production harvest
are sufficient to initiate
germinationofsporesunder sufficient moistureandoxygen.Excludingof
oxygen through vacuum packaging or replacing the container headspace withN2
or C 0 2 adversely effects shelf life. On the other hand, it has been shown that
exclusion of water or its reduction below certain level can prevent germination
in case of B.bassiana, V. Iecanii and M. flavoviride. However,inherent
physiological characteristics offungalspeciesoreven an isolate withina
species, gave distinct survival patterns.

4.2 Hydrophobicity of Fungalspores

The fungal conidia of important fungi suchas Beauveria spp., Metarhizium spp.
and Paecilomyces spp. are extremelyhydrophobic.Therefore, oils are highly
compatible with lipophilic conidia as well as with the insect cuticle-leaf cuticle
target system. Accordingly, formulations as oil flowable (OF) or emulsifiable
suspension(ES)seem to work well. Oil carrier seemstoenhanceconidial
contact with the insect cuticle, thus enhancing the efficacy of entomogenous
fungi. Petroleum based oil carriers stabilize fungal conidia providing good shelf
life evenatelevatedtemperatures, as compared to short shelf life in plant
derived oils. In the latter, presence of short chain fatty acids is known to be
toxic to conidia. However, there are inconsistencies in the literature about the
stability and the use of paraffinic and vegetable oils (Wraight and Carruthers,
1999). Oil formulations are generally designed for ultra low volume (UL) and
undiluted applications. However, often water-diluted applications are required
on vegetable crops and greenhouse applications. Phytotoxicity could also be a
218 Chapter 9

problem with application containing more than 1% (v/v) oil. This situation
requires the use of emulsifiers, which can be toxic to conidia. Hence, proper
screening of emulsifier for conidial longevity is important.
B. bassiana hyphomycete conidia formulation Naturalis"" is based on a
mixture of vegetable oils and vegetable proteins and carbohydrates. In a patent
this combination is claimed to enhance contact between the fungus and the
insect host by acting as anarrestant and feeding stimulant (Wright and Chandler,
1995, 1996).
Schwarz (1995) reported a new patented granular formulation of M.
anisopliae. The granules are produced by growing fungal biomass in liquid
fermentation processed as granules under controlled conditions. The fungal
biomass is separated from the nutrient broth through centrifugation and pelleted
by passing through a rotating screen. The fungal pellets are then dried by slowly
withdrawing water in a fluidized-bed dryer. Gentle and controlled removal of
water induces the cells to enter a 'resting state'. The resultant granulesare
vacuum-sealed in plastic to retain viability and purity. The productshows
acceptable storage stability (noappreciable loss of viability foratleast 12
months) under refrigeration. The formulation consists solely of fungal mycelium
and contains no extra carbon source that might stimulate the growth of soil
microflora antagonistic to M . animpline after application. However, ,It was
reported that some virulent M . animpline isolates could not be formulated as
granules.
The various salient features and requisites of mycoinsecticide formulations
are given in Figure 9.4.

Nutritional manipulation ~~~~~l on field very


oxygen and water 4 of conidia half life
(essentialcomponents)

i prevents
protectant

germination
Conidia carriers oil
compatible
with
enhance

of spores wability
I formulated

Figure 9.4 Salient features of rnycoinsecticidesformulation.


Mycoinsecticides 219

4.3 Post-Application Longevity of Fungal Spores

The half-life of a population of conidia directly exposed to full sunlight is a


matter of hours. In casethe target insect inhabits the undersidesof leaves, B.
bassiana GHAconidial half-life canbeextended to severaldays. Numuraea
rileyii conidia on bean and cabbage were found to have a half-life of 3.6 hours
on a sunny day, but when sunlight was physically filtered, it was extended to
over 40 hrs. (Fragues et al., 1988). Several UV protectants can be used as an
adjuvant in the formulationofamycoinsecticide to enhancetheconidial
viability.

4.4 Comntercial
Mycoirrsecticides

Several
entomopathogenic
fungi
now
are
available
commetrcial
as
mycoinsecticides (Table 9.1). Beauveria bassiana can be mass-produced by a
fermentation process and formulated to enable the fungus to withstand UV light
and temperature and humidityextremescommonlyencountered in the field.
MycotechCorporation has productsbasedonGHAstrain of B.bassiana
(Mycotrol@and BotaniGard@) as wettable powder and oil-based formulations for
control of white flies and other insects. (Jaronski, 1997). Troy Bioscience has
registered amycoinsecticideproduct Naturalis@ basedonstrainJW-1 of B.
bassiana, forcontrolof white fliesandother insects (WrightandChandler,
1996).
BioBlast@ is aformulationof Metarhiziumanisopliae fortermite
controlbyEcoScienceCorporation.Inanotherformulation,BioPath@,this
fungus has been usedin bait stations for control of cockroaches, although isit no
longer beingmanufactured(Miller,1995).Bayerobtainedregistrationofa
pelleted dry mycelium formulationof M. anisopliae as BIO 1020@for control of
black vine weevil (Otiorhynchttssulcatus), a major problemon several
ornamental crops in glass houses and nursery stock.
Among the principal fungal candidates studied as insect vector control
agents include Lagenidium giganteum, Culicinomyces clavosporus and species
of the genus Coelomomyces (Federici, 1995). Onefungalmosquitolarvicide
basedona water-borne fungus Lagenidiumgiganteum (Laginex@),hasbeen
commercialized by
AgraQuest,California
a based
company. Thefungal
zoospores infect larvae of all species of mosquitoes buthave been claimed to be
especially effective againstCulex spp.
220 Chapter 9

Table 9.1 Commercial


mycoinsecticidal
products.

Trade Name Organism Target Producer

Mycotrol Beauveria Whitefly, aphids, thrips, Mycotech Corp.,


(For field crops) bassiana mealy bugs, leaf hoppers Butte, MT, U.S.A.
BotaniGard GHA
and
leaf
feeding
insects
(For green house)

ESC 170 GH B. bassiana EcoScience,


ESC 170 Worcester, MA,
U.S.A.

Naturalis-0 B. bassiana White flies and other


Troy
Bioscience,
JW- 1 insects in field
and
green
Phoenix, AZ,
U.S.A. house

Metarhizium Sugarcane
pests
Mycotech
and Corp.,
anisopliae Aenalamia varia U.S.A.
MT,
Butte,

BioPath Metarhizium cockroach


German
EcoScience,
BioBlast MA, Worcester,
anisopliae Termites
ESF 1 U.S.A.

BIO 1020 Metarhizium weevil


Black
vine
Bayer
AG
anisopliae
(Otiorhynchus sulcatus), Germany
citrus root weevil and
termites

PFR97 faecilomyces Whiteflies, spider mites, Thermo Trilogy,


furnosoroseus aphids and diamondback Columbia, MD,
Apopka 97 moth in ornamentals
and
U.S.A.
vegetables

Vertalec Verticillium Lyle,


Whitefly,
and
Tate
aphid
lecanii UK

Laginex Lagenidium AgraQuest,


Mosquito
larvae Inc.,
giganteum CA, Davies,
U.S.A.
Mycoinsecticides 22 1

5 Concluding Remarks
An ideal biocontrol system would be an inexpensive inoculant that could be
applied once or infrequently and sustain itself in the agro-ecosystem. It would
involve a set of technologies to create a fungal product that can be thought of
and used as a broad spectrum insecticide, having the performance and handling
characteristics
classical
of chemical
products,
while
retaining
the
environmentallyfavorablecharacteristics of a productbased on a naturally
occurring microorganism. This in turn, would require addressing of such key
issues as manufacturing,shelf life underunfavorableconditions,ease of
application and efficacy (Miller,1995). Increase in speed of kill and reduction in
moisture threshold forsporegerminationthroughgeneticandphysiological
engineering of fungal pathogen would need to be investigated further. Studiesof
synergism between fungal pathogens and low doses of chemical insecticides
also indicate considerable potential for integrated control applications.

References

1. Bartlett, M.C.and S. T. Jeronski,1988.Massproduction of entomogenous


fungi for biological control of insects,in Fungi 111 Biological Control Systems,
M. N.Burge, ed., Manchester University Press, Menchester, pp. 61-85.
2. Burges, H. D.,1998.Formulationofmycoinsecticides, in Formulation of
MicrobialBiopesticides: Beneficial Microorganisms. Nenratodes and Seed
Treatments, H. D. Burges, ed., Kluwer, Dordrecht, The Netherlands, pp. 131-
183.
3. Charnley,A.
K.,
B.
Cobb
and J. M. Clarkson, 1997.Towards the
improvement of fungalinsecticides, in MicrobialInsecticides:Novelty or
Necessity?, Symposium proceedings No. 68, British Crop Protection Council,
Famham, pp. 1 15-1 26.
4. Fargues. F., M. Rougier, R. Goujet and B. Itier, 1988. Effect ofsunlighton
field persistence of conidia of the entomopathogenic hyphomycete Narnuraea
rileyi, Entomophaga, 83(3), 357-370.
5. Federici,B. A,, 1995.Thefuture of microbialpesticidesasvectorcontrol
agents. Jour. Anrer. Mosq. Con. Assn., 1 l(Part 2), 260-268.
6. Gardner, W. A.and C. W. McCoy,1992.Insecticidcsandherbicides, in
Biotechnology of Filamentous Fungi: Technology and Products, D.B.
FinkelsteinandC.Ball, eds.,Butterworth-Heinemann,Stoneham, MA, pp.
335-359.
7. Guillon,M.,
1997.
Production of biopesticides:scale up and quality
assurance, in Microbial Insecticides: Novelty or Necessity?, Symposium
Proceedings No. 68, British Crop Protection Council, Famham, pp. 151-162.
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T. M. Odelland D. R. Smitley,1996.Hostrange of thegypsymoth
(Lepidoptera: Lymantriidae) pathogen Entonrophaga nraunaiga (Zygomycetes:
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709-72 1.
9 Jaronski, S. T., 1997.Newparadigms in formulatingmycoinsecticides, in
Pesticrde Fornlulations and Applicatior~ Systew, Vol. 17, G.R.Goss, M. .J.
Hopkinson and H. M. Collins, eds., American Society for Testing of Materials,
pp. 99- 1 12.
IO. Lane, B. S., A. P.Trinci and A. T. Gillespie,1991.Influenceofcultural
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Protozoa urd Fungi, C.M. Ignoffo, ed., CRC Press, Boca Raton, FL, pp 15 1-
236.
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in Bioratiorlal Pest Control Agents: Fortnulatior1 and Delivery, F.R. Hall and
J.W. Barry, eds., American Chemical Society, Washington, DC, pp.213-220.
13. Schwarz,M.R.,1995. Metarkiziun~anisopliaeforsoilpestcontrol, in
Bioratiorlal Pest Control Agents: Fortnulation and Delivery, F.R. Hall and J. W.
Barry, eds., American Chemical Society, Washmgton DC, pp. 183-196.
14. St Leger, R. J., P. K. Durrands, A. K. Charnley and R. M. Cooper, 1988. Role
of extracellular chymoelastase in the virulence of Metarhizizom anisopliae for
Manduca sexta, J. Invertebr. Pathol., 52, 285-293.
15. Wraight, S. P., and R. 1. Carruthers.1999.Production.deliveryanduse of
mycoinsecticides for control of insect pests on field crops, in Biopesticides:
Use and Delivery, F.R. Hall and J.J. Menn, eds., Humana Press, Totowa, NJ,
pp. 233-269.
16. Wright, J. E., and L. D. Chandler, 1996. NaturalisTM, a biopesticide(Beauveria
bassiar~aJW-I) forcontrol of economic Insectsinfield crops,vegetables,
ornamentals and greenhouses, with emphasis on control of Bemisia, Proc. X X
Int I. Cong. Entomol., Firerue, Italy, p. 698.
17. Wright, J. E., and L.D. Chandler, 1995. Biopesticide composition and process
for controlling insect pests, U.S. Patent 5413784.
Part 111

Integrated Use and Commercialization


of Biopesticides
This Page Intentionally Left Blank
IO
Integrated Use of Biopesticides
and Synthetic Chemical Pesticides

1 Introduction

Adverseenvironmentalimpact,increasinginstancesofpest resistance and


public concern about food safety are among the major driving forces behind the
multifaceted efforts towards the development of biopesticides. These include
bioinsecticides for controlof insect pests of agriculture andpublichealth
concern,biofungicides for the controlofplantdiseases on agronomicand
horticultural crops, andbioherbicides for controlofweeds.Several scientific
groups searching for biological means for control have addressed a number of
significant pests, fungaldiseasesandweeds.Theprimaryconsiderations in
developing such products have been a) cost effectiveness, b) user friendliness
and c) adaptation with existing pest control programmes andd) their consistency
and efficacy.
Since the beginning of pesticide use for agricultural purposes,
combinationsoftwoormorechemicalsforsequentialorsimultaneous
application on a single crop have been routinely used. Often, these combinations
produced interactions with either increasedordecreased efficacy, termed as
synergistic orantagonistic effects. Synergisticapproachhasbeenwidely
recommended as a resistance management strategy. Synergistic action occurs,in

225
226 Chapter 10

broadterms,where the activity ofacombination is muchgreaterthan that


expectedfrom their individual effects. Acombinationofbiopesticideand
synthetic chemical pesticide can also result in reduction in quantity of the highly
toxic chemical, and/or reductionin the cost of pest controlapplication.

2 Integrated Use of Biofungicides


Biological
control
systems of cropsagainst
phytopathogenic
fungi are
essentially usingoneorganism(thebiocontrolagent) to controlanother
organism (the pathogen). Biological control systems can be developed as an
alternative to fungicides or for integrated use in combination with fungicides.
Apparently, the integratedapproach is more reliable as biologicalcontrol
systems on their own are subjected to widely varying environmental influences,
which in turn affect their efficacy. Indeed,biocontrolsystemshavebeen far
more successful with protected crops and post-harvest control where there is a
degree of controlon environmental parameters.
A desirable objective of employing a mixture of two or more pesticides
is tohave synergistic action. Advantagesof synergistic combinationsmight
include utilization of a lower dose rate due to increased efficiency, a broader
spectrum of activity and reduced risk of resistance development. Fungicides are
widely used as combination of two or more active ingredients. A formulated
fungicide mixture often contains the 'at risk' fungicide in combination with a
partner with a different mode of action. This is one of the practiced methods of
delaying the onset of resistance, which include, use of fungicide combination
formulations and substitution of a dissimilar material with distinctly different
mode of action for every other application. In fungi-toxic combination products,
to alleviate the risk of fungal resistance due to single site fungicides, mixture
partners are often non-systemic contact fungicides that operate via a multi-site
mode ofaction.

2. I Synergistic Combination of Bacterial Antagonists

Similar to fungi-toxiccombinationproducts, integration ofmorethanone


bacterial antagonistsmayprovidemore effective biocontrolthan the useof
individualantagonists on their own.Enhancedbiologicalcontrol of plant
pathogens using two or more antagonists has been experimentally demonstrated
for anumberofpathosystems.Specificcombinations of different strains of
fluorescent Pseudomonads were shown to suppress take-all disease of wheat
infested with G. graminis ver. Tritici, whereas strains used individually did not
give
comparable
control (Pierson and Weller, 1994). Similarly, it was
demonstrated that postharvest decay of apples caused by Botrytis cinerea and
Use Integrated of Biopesticides 227

Penicillium expansum was controlled by a combination of Acremonium breve


and Pseudomonas spp. (Janisiewicz, 1988).
A number of Bacillus spp. have beentested and found tobe suitable for
development as biofungicidesagainstphytopathogenic fungi. Theseinclude
Bacillus brevis, B. polymyxa, B. cereus, B. licheniformis and B. subtilis. Some of
these are antagonistic to both mycelial growth and conidial germination, whilst
others are antagonistic to only one or the other of these development stages. A
Bacillus antagonisttoonestageofdevelopmentof Bofrytis (e.g.conidial
germination)might be combinedwithanantagonist to anotherstageof
development (e.g. mycelial growth) to provide more effective control of grey
mold. However, for integrated disease control using a combination of Bacillus
antagonists, it is important to know the modes and mechanisms of biocontrol,in
order that additive effects may be achieved. For example, some combinations
such as, B. licheniformis and B. polymyxa showed inhibition of each other in
vitro studies, and should be avoidedas a combination (Seddon etal., 1996).

2.2 Synergistic Combination of Fungal and Bacterial Antagonists

A unique combination of fungal antagonist Gliocladium virens and a bacterial


antagonist Burkholderia cepacia wasreported to behighly effective in
controlling several serious soil-borne diseases of corn, tomato, and pepper in
greenhouse and field tests (Lumsden, 1999; Mao et al., 1998a)). The diseases
(and respective pathogens) controlled include damping-off, root rot, and stalk rot
of corn in the field; seed rot and damping-off (caused by Pythium ultimum and
Rhizoctonia solani) and Southern blight (Sclerotium rolfsii) on both tomato and
pepper plants, as well as Fusarium wilt (Fusarium oxysporum) of tomato and
Phytophthora blight (Phytophfhora capsici) of
pepper.
Thebiocontrol
treatments were applied as seed treatments and root drenches. The biocontrol
treatments significantly reduced disease, as measuredbyplant stand, disease
severity, plant fresh weight,and fruit yield, bothwhenappliedaloneor in
combination. The combination treatment resulted in improvements by reduced
disease severity and enhanced fresh weight for pepper and fruit yield for tomato
in the field, compared to that of either antagonist when appliedalone.
Similar use of naturally occurring, beneficial fungi and bacteria has
beenmadetoprotectcornseedfromdiseasecausedby the fimgalplant
pathogens Pythium and Fusarium (Ma0 et ai., 1998b). In one field study, only
about half the seeds sprouted and grew to mature plants in plots harboring both
fungi. But in plots where seeds were coated with a combination of beneficial
fungiand bacteria, morethan 80 percentbecamefull-grown plants. This
bettered or equaled the performance of seed protected with coatings of any of
several fungicides. Protection by the good microbes continues after the seedling
stage. Mature plants in the biocontrol plots hadabout 25 to 40 percent less
228 Chapter 10

damagefromrootand stalk rot diseases, comparedtoplantsgrownfrom


untreated seed.
Enlisting multiple species of beneficial microorganisms is a new form
of biological control. In the past, the approach has been to use one biocontrol
agent against one plant pathogen. Butthis typically does not guard against other
disease-causingorganisms that mayalsobe inthe soil. Advancementsand
improvements have also been made in the area of shelf-stable formulations and
effective delivery systems for this combination biocontrol treatment (Lumsden,
1999).

2.3 SynergisticInteractionswithFungicides

Several reports have appeared stating the synergistic phenomenon involved in


the integratedcontrolusingfungicidesandfungalantagonistsmaybemore
efficient and longer lasting than the control achieved through fungal antagonists
orfungicidesalone.However for successful integration ofbiologicaland
chemical control of plant pathogens, the system must be compatible. In several
cases, integration of biocontrol agents with fungicides has been shown to be
beneficial reducing the use of fungicides in the agricultural environment (Table
10.1).
Use
of Trichodermaharzianum T
39 (Trichodex)
alone
or in
combination with fungicides has been reported for control of Botrytis diseases in
greenhousecropsand in vineyards(Eladet al., 1994).Alternationwith
fungicidesresulted in as gooddiseasesuppression as that achievedby the
fungicide alone and more consistent than that by biocontrol alone. This approach
hasbeen tested on tomatoandstrawberry for controlof B.cinerea with
minimum number of chemical sprays. Similar observations were made in the
controlof Bottytis bunch rot ofgrapeusing T. harzianum andhalf rates of
dicarboximide fungicide iprodione which provided extremely effective control
(Harman et al., 1996).
Significant synergistic interactions with fungitoxic compounds (which
are as much as 50 times as active as individual enzyme) have been reported
(Lorito et al., 1994). For example, a fungi-toxic sterol, miconazole was needed
in 300 ppb for achieving 50% control (ED,,) of B. cinerea. The addition of 10
ppm endochitinase enzyme from T. harzianum reduced the amount needed of
miconazole to only 70 ppb. The mode of synergistic action postulated is that
chitinolytic enzymes weaken the cell wall of the target pathogen and
consequently facilitate uptake of fungitoxic compound.
Integrated treatment of cottonseed with Gliocladium virens at reduced
levels of metalaxylfungicide is reported to result in a synergistic action
(Howell, 1991). The suppressionof Pythium ultimum damping-off disease of
Use Integrated of Biopesticides 229

Table 10.1 Synergistic fungal antagonists and chemicalfungicidecombinations

Disease Fungal Chemical Applicatiorl


(Target Pafhogen) Antagonist Fungicide Mode

Botrytis diseases in green


house Trichodertna Different Alternated
and in vine yards,suchas,grey Aarzianum T39fungicides
mold in tomato and strawberry(Trichodex)
(B. cinerea)

Crown apple
ofrot
and root Enterobacter Metalaxyl
Alternated
trees (Phytophthora cactorutn ) aerogenes B8

Botrytis diseases Bacillus Dicarboximidcs


(B. cinerea) antagonists such as
such as B. vinclozolin and
brevis iprodione
Damping-off of cotton
seedling Gliocladiunt Metalaxyl Combination
virerrs

Fusariunl root rot disease and Bacillus Combination


Vitaflo-280
pre emergence damping off of subtilis (A combination
lentil (Fusarlunl avanaceum) formulation of
carboxin and
thiram

cottonseedling achieved was found to be equal to fill strength fungicide


treatment.
Crown and root rot of fruit trees is primarily caused by Phytophthora
cactorum. The application of Enterobacter aerogenes (B 8 strain) as a soil and
trunk drench reduces the percentage of apple trees with crown rot infection.
Alternated application of E. aerogenes and metalaxyl is reported to significantly
reduce infection of P. cactorum and increase in fruit yield (Utkhede and Smith,
1993).
Dicarboximides (vinclozolin and iprodione) reportedly show nil or low-
level effects against Bacillus antagonists and are best suited for integration with
them (Seddon et. al., 1996). It is of interest to note that gramicidins, produced by
Bacillus spp. such as B. brevis and the dicarboximides, both act on membranes
albeit by different suggested mechanisms of action. It has been proposed that
dicarboximides act on membranes by free radical reactions with primary effects
on lipid peroxidation. On the other hand, gramicidins are thought to act by
disrupting the integrity of membranes by interaction with the phospholipids. It is
230 Chapter 10

therefore, likely that the combined action of gramicidins and dicarboximides is


greaterthanthese two componentsactingaloneinthatgrarnicidinsmight
provide easier access for the dicarboximide to the membrane lipids, and thereby
more effective peroxidation.
Bacillus subtilis strain GB03 suppresses pathogenic Fusarium spp. and
R. solani.Itisreported that B. subtilisGB03 in combinationwithstandard
chemical fungicides gave improved results from R. solani inoculated plots. The
successof B. subtilisGB03 in the cottonmarket is attributed due to its
integration with standard chemical fungicides. It is postulated that B. subtilis
GB03 supplementsstandardchemicalfungicidesthrough an earlysynergy,
expandstheactivityspectrumandprovideslong-termactivity(Brannenand
Kenney, 1997). A combination product of B. subtilis GB03 with metalaxyl and
PCNB has been commercialized underthe trade name System 3 Seed Treatment
by Helena Chemical Company(Memphs, TN, USA) for control of Rhizoctonia,
Phytophthoru and Pythium diseases.
Similarly,integrateduse of Bacillussubtilis with Vitaflo-280(a
combinationformulation of carboxin and thiram), is reportedasthe most
effectivemethodforreducingdiseaseseverity due to Fusariumavenaceum,
whichcausedrootrot and pre-emergencedamping-offinlentilseedling
(Hwang,1994)(Figure
10.1). B. subtilis
producesseveral
anti-fungal
compoundsincludingbacilysinandfengmycinwhichareinhibitory to root
pathogens such as F. avenaceum. Competition between F. uvenaceum and B.
subtilisfornutrientsandinfectionsites may alsoplayarole in biological
control.In this particularcase itwas found that Vitaflo-280,athigher
concentration (50 ml I 1) was non-toxic to B. subtilis, whereas it was highly toxic
to F. avenaceum at lower concentrations (1 mlll). Thus the two systems have
potential compatibility for an integrated application for disease control. In effect,
dual B. subtilis and Vitaflo-280 treatments significantly improved the efficacy.
In this integrated system, seeds were better protected and grew more rapidly in
F. avenaceum-infested soil than if they were treated similarly with Vitaflo-280
alone.
with
infested
Soils
Fusarium avenaceum -
(A fungal pathogen)
caused

reduced Integrated
Lentil
seedlings
and
Root rot
with t pre-emergence
damping-off

Treatment with
B. subtilis + Disease compared Vitaflo 280
Vitaflo 280 severity to alone

FigurelO.1 Integratedtreatment of B. stcbfilis and Vitaflo improved disease


control as compared to treatment with Vitaflo only.
Integrated Use of Biopesticides 231

3 Integrated Use of Bioherbicides


A useful scenario would be combining one chemical or herbicide with another
herbicide, in quantities less than those required when either component is used
alone to provide superior and/or more economically advantageous weedcontrol.
Mechanism for this phenomenon includes combination of compoundsthat act at
different molecular sites or that chemicallyblockmetabolicdegradationof
another
compound.Herbicidemixtures fall into two
categories
(often
overlapping).In the first type, eachherbicide is used ata f i l l rate and the
spectrum of weeds controlled by eachis mutually exclusive. In the second type,
both control the same weeds.
Therehavebeensomeanalogous results usingchemical interactions
withbioherbicides that haveprovided beneficial, additiveor synergistic
enhancement of bioherbicide activity. If synergistic interactions are found those
expand the bioherbicide host range, options are available to maximize the host
regulation potential.

3.1 Rationale of Synergistic Combinations

Likely
scenarios
synergistidadditive
of interactions chemicals
of and
bioherbicides for weed control are as follows:
a)Weeddefenses are loweredusingherbicides(oranyotherchemical),
making weeds more susceptibleto pathogen attack;
b)Bioherbicideconcentrationand quantity, chemical(herbicideor other)
concentration, or bothare reduced and
c)Target weed rangeofagivenpathogen is expandedby the use ofa
chemical synergist.

Plant(weed)defenses(physicalkhemical barriers andbiochemical


responses) attempt to protect plants against attack from essentially all micro-
organisms.Pathogensincluding viruses, bacteriaand fungi, possess infection
mechanisms that allowthem to either evadeorbreak down plantdefenses,
causing infection that can lead to injury or death. Herbicides, that by design
causeplant injury, may be the primarychoice for acting as synergistsof
pathogens. According to Gressel et. al. (1996) impermanent synergies such as
using chemical herbicides, may have advantages, as these provide weed control
whenappliedand the level of the organismshould later dissipate, like an
environmentallysoundchemical herbicide. Weedscanevolve resistance to
pathogens just as they have to conventional chemical herbicides. The use of
chemicals as synergists or the synergistic use of two pathogens should delay or
even overcome such an evolution, just as it has with conventional synergists for
chemical pesticides.
232 Chapter 10

3.2 Synergistic
Interactions of Fungal Pathogens and Chemical
Herbicides

The fungal bioherbicides require specific conditions such as a prolonged dew


period or freemoistureforsporegerminationandinfection. Thus variable
environmental conditions may severely hamper effective control of weeds. To
overcome this limitation, integration of fungalbioherbicideswithsynthetic
herbicidal chemicalshave resulted in synergybetween the two approaches.
Compromising the plant in some way with a chemical agent reduces the plant's
ability to mount a defencetopathogenattack,improvingtheefficacy of
bioherbicide.
Hoagland (1996) hasexpanded a compilation of several
synergisticinteractionsbetweenfungalpathogensandherbicides as givenin
Table 10.2.

Table 10.2 Synergisticfungalbioherbicidesandchemicalherbicidecombination


(Adapted from Hoagland,1996 ).

HerbicidePlant Plarlt
Fungal pathogen Growth Repulator

Phytopllthora tmguspenna Glyphosate Soybean


Thidiazuron Velvet leaf (Abutilon
Colletotrichunl coccodes theopkrasti Medic)
Water milfoil (Myriophyllum
Colletotrichum
gloeosporoides
Endothall spicatum L.)
cassiae
Alternaria Glyphosate Sicklepod (Cassie obtusrfolia)
Water hyacinth
Cercosporu rodtnord Diquat (Eichorrlia crassipes (Mart.)
Solms)
Barnyard grass
Cochliobolus
lullatus Atrazine (Echinochloa crusgalli (L.)
Beauv.)
Bentazone and Florida beggarweed
Fusarium laterilium (Disn1odium tortuosurn(SW.))
Acifluorfen

PytAiunl and Fusarium spp.


Glyphosate Blackbean
Fusarium solanif: sp. Trifluralin Texas gourd (Cueurbita texuna
cucurbitae (Scheele) Gray)
solarli
Rhizoctonia Trifluralin Bean
integrated Use of Biopesticides 233

Caulder and Stowell (1988) of Mycogen Corporation received a US.


patent on the use of synergistic interactions of some herbicides and four fungal
pathogens.Theherbicidesacifluorfenandbentazonwere the most effective
synergists, providing increased control of several weed hosts by their respective
bioherbicides,e.g.sicklepodby Alternariacassiae, northernjointvetchby
Collectorichum coccodes, hempsesbania
by C.
truncatum and
Florida
beggarweedby Fusariumlateritium. These interactions also pointedout that
there is no universal chemical synergistfor all pathogens.
Sharonet.al.(1992)studiedbiochemical interaction of Alternaria
cassiae andsicklepod (Cassie obtasifolia). Thepathogencausedanelevated
level of production of a flavonoid phytoalexin in sicklepod, which was found to
be fungi-toxic. Treatment with glyphosate suppressed this defense response of
the weed by
lowering
phytoalexin production.
Thus
glyphosateacted
synergistically with this pathogen, by suppressing weed defenses. Twenty-fold
less glyphosatethan is normallyphytotoxic,suppressed the phytoalexin
production and increasedthe intensity of infection. (Figure 10.2).

3.3 Synergistic
Interactions of Bacterial Pathogens and Chemical
Herbicides

Combinations of bacteria and chemical herbicides for enhanced weed control


termed X-tend bioherbicide systems by Crop Genetics International (Hanover,
MD, USA) have been reported. The herbicide sulfosate (a sulphur analogue of
glyphosate), causes varying degree of plant injury to a wide variety of weeds
such as pigweed, barnyardgrass, yellowfoxtail and johnsongrass. Bacterial

Alternaria cassie Interaction ~ Cassie obtusfolia producedt Fungitoxic


(Bioherbicide)

Glyphosate
y phytA;ld
with (Sicklepod
weed)

i
phytoalexin

A defence response
(subleathal dose) production of the weed

1 enhancing

Biohehicide
adivity

Figure 10.2 Synergy between a fungal pathogen and synthetic herbicide.


234 Chapter 10

strains alone caused little or no plant injury, but sulfosate plus bacteria resulted
in greater injury than from the herbicide alone (Christy et al., 1992). Similar
synergistic results were obtained in field tests of two bacterial preparations with
anotherherbicide glufosinate. Thesynergybetween synthetic herbicidesand
bacterial agents could significantly reduce amounts of herbicides used to control
abroadspectrumofweeds.Nevertheless,improved bacterial strains andor
formulations are needed to make successful commercialisation of such products
for weed control.

4 Integrated Use of Bioinsecticides


In insect control, combinationformulationshavebeenmadeoftenusinga
relatively inexpensive, poorly active compound with more expensive and more
active compounds. Salut for example, a combination formulation
of
chlorpyrifosanddimethoatebyBASF,gave rise toadditiveand synergistic
action controlling a broad range of insects (Neumann et al., 1984).
Similarly, bioinsecticides such as Bt, in combinationwithmany
synthetic insecticides or in sequential application with synthetic insecticides has
displayed synergistic activity.

4.1 Synergistic Combinations of Btk with Chemical Insecticides

4.1.1 Btk with


Enciosuvan

Integrated use of Bacillus thuringiensis subsp. kurstaki (Btk) with endosulfan


has been subject of investigation for the control of bollworms of cotton. An
alternated applicationofa Btk formulation(DiPel 8L) (@ 750 I& ha)and
endosulfan (@ 2.5 1 I ha) on cotton was found to result in reducing the number
of sprays of synthetic insecticide from 6 to 3. The control of bollworm complex
of American cotton (Helicoverpa armigera and Pectinophora gossypiella) was
found effective and comparable to that with the recommended spray schedule of
insecticide, increasing the yield by 52%. Over a 3 year long study, it was also
observed that Btk alone gave an inconsistent performance, and did not prove
better than recommended insecticidal treatment. (Butter et al., 1995). Similarly,
in a laboratory bio-assay, an application of a Btk formulation (DiPel 2X) to
cottonleavesatlowconcentration (LCs) resulted in enhanced activity of
endosulfan and reduced resistance (7-fold to 2-fold) to endosulfan in larvae of
H. armigera (Pree and Daly (1996). The authors suggested that a mixture of Btk
and small quantities of endosulfan might result in increased effectiveness over
Btk alone and serve to reduceresistance to endosulfan (Figure 10.3).
Integrated Useof Biopesticides 235

No. of application
Dipel 0 L (from 6 to 3)
a750 mVha
+ Alternated Cotton
application
Endosulfan increased
@2.5 Vha Yield by 52%

Bollworm complex
(Helicovepa arm'gera,
a Pectinophora gossyplella)

Resistance to
Dipel 2X endosulfan
@LC, conc.
Integrated Cotton J
/reduced + (7-fold to 2-fold)
+ application leaves
Endosulfan
I against Activity

t
H. armgera
b
neonate larvae

Figure 10.3 Synergybetween Bacillus


tkuringiensis subsp. kurstaki and
endosulfan, (a) alternated field applicatlons resulted in enhanced yield of cotton
and reduced number of endosulfan applications, (b) In laboratory bioassay, a
tank-mixapplicationresulted in enhancedactivityandreducedresistanceto
endosulfan.

Cibulsky et al. (1993) have quoted the results of an evaluation of a high


potency Dipel emulsifiable suspension (ES) formulation on cotton in USA and
Australia. The DiPel ES was applied with a spray oil (DC-Tron) and tank mixed
withendosulfananddeltamethrin(Decis)respectively for controlof H.
armigera and H. punctigera populations.Dipel ES in combinationwith
endosulfan resulted in 42% less boll damage, as compared to Dipel ES by itself
and Dipel ES and deltamethnn combination (Table 10.3).
Chapter 10

Table 10.3 Evaluation of DiPel ES for Heliothis control of cotton


(Adapted from Cibulsky et al., 1993).

Spray
Rate volume Boll damage
Integrated System
(BIu"/Ha) (l/ha) &)

DiPel 30 30 6
DiPel+
30Endosulfan 2.0 + 1.5 3.5
Dipel + Deltamethrin 30 2.0 + 1.5 7.3
(a) BIU = Billion International Units

4.1.2 Btk with


thiodicarb

Klein et al. (1995) investigated the role of Btk with ovicides in the management
of the heliothine complex in cotton. A far greater reduction in larval population
was observed with combination of Bt and ovicides than Bt alone. It was also
reported that Bt andthiodicarb(Larvin@)providedbestcontrolofseveral
ovicides tested.

4.1.3 Btk with


Imidacloprid

Btk preparations in combinationwithimidaclopridhavebeenclaimedto


enhance insecticidal activity (Schnorbach,1995).Imidacloprid is asystemic
insecticide and functions as insect nicotinergic acetylcholine receptor (nAChR).
It strongly binds to acetylcholine-receptors on the nerve cells of insect blocking
the binding site of acetylcholine. The resultant interruption of neural
transmission paralyses the insect and it dies. Imidacloprid displays selectivity
between insects and mammals. Even in common receptors that exist in insects
andmammals,adequate levels of selectivity are achieveddueto lesser
sensitivity of receptors in mammals. Compared withrat muscles, insect nicotinic
acetylcholinereceptors are up to 1000timesmore sensitive to the effect of
imidacloprid.Thus, a combinationofimidaclopridand Btk, withthe two
different modes of action, is more effective and yet safer to mammals.

4.1.4 Btk withMycotoxins of Metarhizium anisopliae

A combination of Btk with destruxins (Metarhizium anisopliae mycotoxins) is


reported to demonstrate synergistic interaction at low doses,in a laboratory
Integrated Use of Biopesticides 237

bioassay on the 5' instar larvae of spruce budworm(Choristoneura furniferana).


Based on the dataobtained in the bioassaysinvolvingcombined agents,
extrapolation ofthe model correlating mortality rates as a function ofthe various
lethal doses used for each agent was plotted. It was found that a combination of
two agents at their individual LD20 dose rates would give rise to nearly 55%
mortality. Itwas also observed that acombinationof Btk (at LD15) and
destruxins (at LD40) and vice-versa concentration ratio gave rise to nearly the
same mortality (-72%) indicating that both the agents have similar contribution
to synergism (Brousseau et al., 1998).

4.2 SynergisticCombinations of Bti

4.2.1 Bti with


Teflubenzuron

Chuietal.(1995)evaluated the useof Bti formulation(VectoBac G ) with


teflubenzuron for control of Aedes aegypti larval populations under laboratory
conditions. Individually, both Bti and teflubenzuron were found to be highly
effective in reducing A. aegypti larval population. However, teflubenzuron gave
higher degree of residual toxicity than VectoBac G. Integration of both at their
LC9=,concentrations in different ratios revealed that a 1:9 LC9=, concentration of
Bti and teflubenzuron gave better control i.e. 1.35 times of LCg5of VectoBac
and 1.18 timesof LC9=,of teflubenzuron (Figure 10.4).

Bf-israelensis

Teflubenzuron /
> ~0th
controlled
A. aegyptilarvae
populations

1.35-times of LC% of
Bfi + Teflubenzuron
Bfi and1. I 8-times of
(1:9 LCesconcs.)
LC% of teflubezuron

Figure 10.4 Synergisticinteraction of Bfi andteflubenzuronresult


in improved controlof A . aegvpti.
238 Chapter 10

4.2.2 Bti with


Methoprene

Similarly, Perich et al. (1988) reported laboratory evaluation of a formulation of


Bti combined with methoprene against Anopheles albimanus and An. stephensi.
It was found that Teknar,a Bti formulationalone as well as Duplex,a
formulationconsistingof Bti andmethoprene,ajuvenoid insect growth
regulator, produced 100% mortality in both species. However an expected low
mortality was found in late 4th instar larvae of both species, when treated with
Teknar alone. Significant mortality of late 4th instar larvae and a low adult
emergenceoccurred withboth species in water treated withDuplex.This
demonstrated that Bti canbeused effectively in combinationwithother
biocontrol agents.

4.2.3 Bti withotherBiocontrolAgents

Combination of Bti with mosquito fish (Gambusia affinis) gave better control of
Culex tarsalis coq. populations than when each agent was usedseparately. Neri-
Barbosa et al. (1997) reported that Bti (Bactimos briquettes) combination with
an insect predator species such as the back swimmer Notonectn irrorata uhler
effectively controlled Culex spp. in a cost effective and environmentally safe
manner. They reported efficient reduction of mosquito larvae in both Bactimos
application
alone
and the combined Bactimos
andpredator application.
Nevertheless, from an economical point of view,the combination approach was
found better, as Bactimos had to be added less frequently to containers having
notonectids in them to effect the level of control desired.

4.3 Synergistic Combinations of Baculoviruses with Synthetic Insecticides

A cabbage moth NPV product Mamestrin, has been found effective for control
of its hosts, including H. Zea, H. virescens, H. armigera, S. exiguaand
Diparopsis watersi. On the other hand, Mamestrin, a combination product with
0.3% cypermethrin is claimed to haveanenhancedhostrange.Theadded
speciesinclude S. littoralis, Plusia sp., Plutellaxylostella,Helula unriulis,
Maruca testulalis and Phtorimaea operculella (Cunningham, 1995).
Similar positive interactions between several wild-type baculoviruses
withphotostablepyrethroidshavebeen reported. Aspirotetal.(1987)have
documented potentiation
between Mamestrabrassicae nucleopolyhedrosis
(MbMNPV)anddeltamethrin on Spodopterafrugiperda, S. exigua and H.
virescens. Similarly, MbMNPVwithfenvaleratehasbeenshownto exhibit
potentiation against diamondback moth (Plutella xylostella) (Bianche, 1991). A
significant reduction in the LCso of Pieris brassicae granulovirus (PbGV) was
Use Integrated of Biopesticides 239

observed when combined with a low concentration of permethrin (Peters and


Coaker, 1993).
Shapiro et al. (1994)
observed that Lymantria dispar nucleo-
polyhedrovirus(LdMNPV) in combinationwithNeem extract significantly
reduced the LTSofor the gypsy moth comparedto the LdMNPV-water treatment.
McCutchen et al. (1997) reported positive interactions (decrease in the
median lethal time, LTSo, compared with the LTSOfor either component alone) of
recombinant Autographa californica nuclear polyhedrosis virus (AcAaIT) that
expressesan insect selective neurotoxin(AaIT),whencombinedwithlow
concentrationsof
several insecticides. Cypermethrin and
methomyl were
synergistic in combinationwithAcAaITagainst Heliothisvirescens. On the
other hand, another recombinant virus, AcJHE.KK, that expresses a modified
version of juvenile hormoneesterase, showed no evidence ofeither synergism or
antagonism in combinationwithcypermethrin.Inanotherstudy, the results
indicate that the pyrerhroids did not enhance speed of kill indiscriminately with
any recombinant baculovirus.
American Cyanamid has patented combinations of recombinant insect
viruses such as AcMNPV.AaIT and AcMNPV. egt- [containing a deletion in the
geneencoding
ecdysteroidUDP-glucosyl transferase (egt)] withseveral
chemical and biological insecticides claiming enhanced insect control (Black et
al., 1996).Theyobservedsynergismwith the recombinantAaIT virus in
combination
with the formamidine, arylpyrrole, diacylhydrazine and
cypermethrin when tested against H. zea larvae. In contrast, with recombinant
virus AcMNPV.egt-, only diacylhydrazine significantly hastened the speed of
kill. However,against H. virescens larvae, both the recombinant virus and
cypermethrin mixtures producedsynergistic interactions.

4.4 Synergistic combination of mycoinsecticides with synthetic insecticides

Entomopathogenicfungimay be usedwithchemical insecticides to obtaina


synergistic or additive action. For example, teflubenzuron, an insecticide which
selectively interrupts chitin synthesis of insects - but not fungi - synergized
mortality of third-instar larvae of desert locusts with M. flavoviride (Joshi et al.,
1992). Similarly, it is reported that the combinations of either B. bassiana or M.
anisopliae ( lo6 to lo7 conididml) with sublethal doses (100 ppm or greater) of
imidacloprid increased synergistically mortality and mycosis of 1st instars of
citrus rootweevil Diaprepesabbreviatus (QuintelaandMcCoy,1997).The
larvae of citrus root weevil are of significant economic importance because of
the severe root injury they cause to the citrus tree. The lethal time was also
reduced significantly through the synergistic interaction of fungaVchemica1
treatment.
240 Chapter 10

A synergisticcombination of buprofezin, and B. bassiana hasbeen


patented which is claimed to be highly effective against a broad spectrum of
different insects including scale insects, whitefly, leafhoppers and thrips (Knauf
and Morales, 1999). The two components can be applied simultaneously or in
succession with the advantage of long residence time and ofuse mycoinsecticide
at a considerably low cost.

5 Concluding Remarks
Itappearsunlikely,thatbiologicalcontrolswillcompletelysubstitutefor
chemical controls, at least in the immediate future. Therefore, the integrated use
ofbiologicalcontrolagentswithexistingpestcontrolstrategieswithin
individualcroppingsystemsmay be a more viableapproach.Alternatingor
combining biological and chemical strategies may increase efficacy of insect
pest, weed and disease control and possibly prolong the efficacy of chemical
pesticides by avoidingorretarding,thedevelopmentofresistanceintarget
organisms. In addition,integration of biologicalcontrolstrategieswithcrop
managementpractices,suchas the manipulationofphysicalchemicalsoil
conditions to favor biological control agents would promote their efficacy and
simultaneously, increase the sensitivity of the pathogen towards the agents.
Coexistence and interaction of microorganisms is the norm in nature.
Due toco-operationamongseveralpopulations of microorganisms,soil
suppressiveness usually limits disease incidence efficiently and consistently. On
the contrary, biological control based on the application of single antagonistic
strain is often inconsistent. One way to improve efficacy and consistency of
biologicalcontrolwould be to mimic thecomplexityofthemechanisms
operating in suppressive soils, and to use several populations of antagonistic
microorganisms together.
With
mixed biocontrol inoculum, potentially
increased diversity of antibiotics being produced and nutrients being utilised
could greatly increase the effectiveness of the product. It has been demonstrated,
forexample, that
co-inoculation of
fluorescent Pseudomonas with root
colonizing fungi always resulted in greater and more consistent suppression of
disease than the application of single microorganism (Leeman et 1996). al.,

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inoculation of fluorescent Pseudomonas spp. and a root colonizing fungi, Eur.
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29. Neri-Barbosa, J. F., H. Quiroz-Martinez, M. L. Rodrignez-Tovar, L. 0. Tejada
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30. Neumann, U. and V. Harris, 1984. BAS 270 001-A versatile broad spectrum
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33. Pierson, E.A. and D. M.Weller,1994. Useof mixturesoffluorescent
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35. Quintela, E. D.andC. W. McCoy,1997.Pathogenicityenhancementof
Metarhizium anisopliae and Beauveria bassiana in first instarof Diaprepes
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Commercialization of
Biopesticides

1 Introduction
Currentgrowth in the developmentofbiopesticides is bothregulatoryand
technology driven. In the late 197Os, there was a growing awareness of the real
and potential problems that synthetic chemical pesticides posedto humanhealth
and the environment as awhole.With this awareness,camemorerigorous
registration requirements that resulted into registration offewernew active
ingredients. Simultaneously, the averagedevelopmentcostperproductgot
increased to $ 50 million. The problem has been compounded by the fact that
pests became resistant to many pesticides, resulting in abandonmentof the
product or the need for higher application rates. On the other hand, improved
understandingofbiologicalprocessesandprogress in geneticengineering
techniques have given a new impetus to developmentbiopesticides.
of
In the United States, regulatoryandtechnologydrivengrowthof
biologicalpestcontrol alternatives is especiallypronounced.Inorderto
strengthenandaccelerate the EnvironmentProtectionAgencys pesticide
registration program, the U. S. Congressamended the federal insecticides,
fungicides and rodenticides actin 1988. Because ofthe costs associated with the
re-registration process, many pesticide companiesdropped the registration of
products for the small markets. This causedsignificant problem to small market
245
246 Chapter 11

growers.These factors combinedwith the increasing difficulty and costs to


develop and register new classes of chemical pesticides, have encouraged the
search for safe alternatives for control of plant pests (WilsonandJackson,
1997).

1.1 Fast-trackregistration f o r biologically-basedtechnologies

In recognition ofthe need for pest control alternatives to chemical pesticides, the
pesticide regulation authorities in many countries have the mandate to put the
development of biologically based technologies for the control of plant pests on
the 'fast track' for registration. The U.S. EPA has recognized that biochemical
and microbialpesticides are distinguished from standard chemical pesticides and
has established different data requirements as part of its registration regulations.
Biopesticides currently are subject to a three-tier toxicology testing procedure
and a four-tier environmental testing procedure. Tier 1 testing for EPA requires
data on non-target organisms and environmental fate. A biopesticide product
which satisfactorily completesboth the Tier I toxicologyandenvironmental
tests is not required to go through the tests specified in subsequent tiers.
Registration authorities treat each case on its merits and according to
the purposeof the programand the target pathogen,and will select the
evaluation process to meet the nature of application. For instance, biofungicides
for post-harvest decay control in packing houses were relieved of the need to
conduct ecological testing, since the organisms are expected to be applied only
in aconfinedenvironment(HofsteinandChapple,1999).However,should
questions arise during any tier of testing, additional tests may be required. This
translates into less regulatory requirements for a biologically-based system than
a chemical pesticide.
Thus, as againstmorethan120 safety andother issues related
documents to support the registration of a chemical pesticide, current US EPA
requirements for a biological fungicide, for example, are for approximately a
dozen studies. This difference means huge cost and time savings in favor of the
biologicals. Whereas the registration cost of a chemicalpesticide might be $ 10-
20 millionandtake 5-8 years, the registration cost of a biological pesticide
might be 1/10" or less of that figure and the time period may be only 1-2 years.
The h g h cost, long-term safety studies such as carcinogenicity, plant and animal
residue and ecological fate and effects are not required forthe biological control
agents (Froyd, 1997). For a biopesticide product completing only Tier I testing,
approximately one year of laboratory testing is required. Subsequent U.S. EPA
registration generallytakesapproximatelyone year. Toxicology testing, field
development trials and related costs for U S . EPA registrations incurredfor
biopesticides have averaged under$500,000 for each productregistration.
Commercialization of Biopesticides 247

1.2 Genetically engineered microorganisms for pest control

In the US, regulatory authorities have taken the approach that no distinctions
would be made between natural and genetically altered organisms in terms of
the regulatory requirements to prove safety and efficacy. In July 1990, The US
approach to regulate biopesticides was further laid down in the 'Principles for
federal oversight of biotechnology: Planned introduction into the environment of
organisms with modified hereditary traits'. This underlined the position that no
distinctions between natural and genetically altered organisms would be made,
focusinginstead on characteristics oforganismandenvironmentconcerned.
Thiswasbasedona set of scientific principles for the evaluation of
biopesticides, including the characterization of organisms, the spread into the
environment, as well as effects on humans, domestic animals, wild life and non-
target organisms. The first genetically engineered products approved under the
new US legislation were Mycogen's biopesticides MVP@ and "Trek@
(discussed in Chapter 3).
Themajorchallengewithdevelopmentandcommercialization of
genetically engineered biopesticides is the production of an effective product
that poses little or no risk to health or the environment. These concernsinclude,
(a) the release of any engineered organism into the environment raises
questions ofpotential effects to human health and non-target organisms;
(b) that engineered organisms will displace naturally occurring organisms
from their niches, thereby causing ecological perturbations;
(c) if an engineered organism exhibits unanticipated and deleterious
environmental properties, it may be difficult to eliminate the organism
from the environment andlast, but not the least,
(d) the foreign geneticmaterial
may be transferred
from the released
organism to other organisms with unpredicted consequences.

These issues need be satisfactorily addressed prior to the field release


of genetically engineered microorganisms such as baculoviruses (Wood, 1995).
There
are also functional requisites touching upon
mass
production,
effectiveness and stability for successful commercialization.

2 Commercialization Aspects
A commercial biopesticide is defined as one which works consistently in a field
environment and that can be economically produced and formulated in a form
that will maintain the viability of the organism through commercial distribution
system. In order to be successhl for commercial application, biopesticides must
be efficient, dependable, cost-effective and safe for humans, the crop and the
environment.
248 Chapter 11

The U S . CongresshasenactedtheFoodQualityProtectionAct
(FQPA)1996,whichremoved the DelaneyClause related to cancercausing
pesticides that appear in processed foods. At the same time, it mandated greater
margins of safety for all new pesticide tolerances. It requires that all U.S. EPA
tolerances be reassessed using new standards within10 years. All tolerances will
now be based on a "reasonablecertainty of no harm" and there will be a specific
determination ofrisk to infants and children.
Thereissomeexpectation that duetonew risk requirementsmany
older products may have to be dropped. If this were to happen, the viability of
biocontrolsshould increase, as there wouldbefewercheaperchemicals to
compete with. Although the investment differentials exist in the development of
biologicalsandchemical pesticides, the priority of the industry is market
potential and profits. Thereforeonlythosemicrobialsshowingpromise of
providing an economic return will be developed as biopesticides. In fact impetus
for development of biopesticidesmaybe the existence of significant pest
problems that can not be solved by chemical pesticides (Gardner and McCoy,
1992).
There is great optimism that biologically-based alternatives can
successfully integrate with, compete with and even replace synthetic pesticides
in certain applications (Froyd, 1997). Even more significant than FQPA as an
agent for change is the rapidintroductionandacceptanceof biopesticides.
Recently most of the newactive ingredients registered in the United States have
been biopesticides, including biochemicals and genetically modified organisms
(GMOs). It is anticipated that some biopesticides will become major tools for
organic farming, integrated pest management (IPM), and as replacements for
products lost due to FQPA (Stewart, 1999).

2.1 TechnicalConstraints of Commercialization

There have beencertain technical barriers that have prevented broad commercial
utilization of biopesticides. Someof the keyareasincludefermentation,
stabilization and
delivery
technology that neededmore
focus for
commercialization to succeed. There is also a general problem that pertains to
basic researchers, who do notunderstandwhat is needed to successfully
commercialize a biological product. This eludes them in focussing their energy
and resources on resolving the key technical issues (Zorner et al., 1993). Also
manyorganismsprovideexcellent results underlaboratoryconditionsbut
currently there is no practical way to produce an economical product from these
organisms. Researchers
need to beaware of industrial production
and
agricultural restraints. Besides,goodlaboratory results do notnecessarily
correlate to good field results. Lab tests are often conducted with biologicalsin a
form that doesnotcloselyresemble that producedthroughfermentation
technology(BrannenandKenney,1997). It is therefore, important that the
Commercialization of Biopesticides 249

individual scientists make liaisons with people who have these skills and thus
work in a multi-disciplinary environment
In 1996, the Society for Industrial Microbiology in the US. organized
twosymposia on the formulation and commercialization of biological based
products for the control of plant pests. In the symposia, various technological
constraints that are shared by living microbial biocontrol agents and that have
hindered their commercial use forthe control of insects, weeds andplant
pathogens, were identified. These included (a) the lack of availability of low
cost production methods, (b) the need for development of suitable microbial
formulations with reasonable shelf lives, (c) the need for consistent pest control
under field conditions and (d) the need to ensure that the plant pathogens that
form the base of the commercial pest control products do not pose an
environmental threat to other plants or segments of the eco-system in which they
will be used (Wilson and Jackson, 1997).

2.2 Market Environment

Biologicals have been unable to penetrate beyond niche markets so far, and even
this role is threatened as a new generation of pesticide chemistries has come on
line andtransgenic plants reduce the size of the sprayable pesticide market.
Small niche markets tolerate the mediocre performance of biopesticides due to
lack of competition and as a trade-off for their environmental and user safety.
However this does not amount to a marketing opportunity but user acceptance of
weakly performing product(s) within these technology-starved markets (Stowell,
1993). It is imperative that performance standards of biopesticides are raised to
improve their commercialization prospects.
Various factors necessary for commercialization of biopesticides
include improvement of product efficacy by technology improvements,
enhancement in their price/performance characteristics and
improved
understanding on how to best use often in conjunction with other products.
Inter-product competition between existing broad-spectrum chemical pesticides,
new selectivechemistries (such asSpinosad for cotton and imidacloprid for
potatoes in competition with Bt), transgenics and novel technologies is
intensifying for maximum market share. This in turn, wouldintensify the
competitive pricing environment and result in rapid shift in individual products
market share. New chemistry products and transgenics would also spur the
adoption of biopesticides through the expansion of the selective controls market
(Shimoda, 1997).
The North American biopesticides market consists of a dual structure;
large diversified companies and small-dedicated companies, which account for a
significant shareof the innovations. Theexistenceofthe small companies
depends on their ability to protect the fruits of their research and signal their
250 Chapter 11

existence to the market. Indeed many small companies focus


on
the
development of new patentable products and processes with the intention of
selling the patents or their sharesto larger firms.Capitalization on market
opportunities and reduction of the business risk tied to a market place, favors
strategic action by smallcompanies to either a) developcritical mass, by
acquisitions and mergers, b) have product(s) with huge market potential, or c)
enter into strategic alliances/ virtual integration with larger companies
(Shimoda, 1997).

2.3 Search for a Suitable Model and Viable Approach

The current approach to commercializing biopesticides is based on a chemical


pesticide model. This paradigm emphasizes major crops and is based on cheap,
stable products that are easy to scale-up and use. Apparently biological agentsfit
the chemical model poorly. Thus a change in approach is necessary, one based
on the realities of biological systems. Thisapproach might emulatebusiness
sectors that have successfully overcome biological limitations. A typical
example is food industry. It has demonstrated that low stability products are not
a fatal flaw. Similarly, the microbrewery industry has demonstrated that there is
a strong market for fresh product soldwithout preservatives. The keys have been
local small batch custom production and quick turnover leading to high quality
products. The concept might be adapted to biopesticides (Gaugler, 1997).
Aconference to review the progress onbiologicalcontrol was
organized by Boyce Thompson Institute and US Department of agriculture at the
Cornel1 University in April 1996. Subsequently, a conference on Biopesticides
and transgenic plants - new technologies to improve efficacy,safetyand
profitability was organized in January 1997 at Washington, DC. The conference
deliberated on interdisciplinary analysis of commercialization challenges. This
was followed by a workshop on the Alternative paradigms for commercializing
biological control at Rutgers University in May-June 1998.This workshop
focussed attention on creating a new mindset for developingand using
biological control and fostering close cooperation among industry, researchers
and extension personnel interested in biocontrol. The furious activity and debate
on the search of most suitable approach for commercialization of biopesticides
continues in many partsof the world.

3 Commercialization of Biofungicides
Commercialization of biofungicides has received a significant boost in recent
years. This has been primarily due to the impressive progress in isolation and
characterization of novel strains of microorganisms that have met basic
Commercialization of Biopesticides 251

requisites for commercialization. The desirables include consistent suppression


ofpathogensunder field conditionsandeasymassproduction in standard
fermentation facilities. Thevarious steps involved in developmentofa cost-
effective biofungicide are in quite rigid order and are critical for their successful
commercialization. These include, isolation of active strain against specifichon-
specific pests, developmentofbioassays,laboratoryevaluations,laboratory
scale-up
production,formulations, field evaluations, product registration,
commercial production and market acceptance of new technology (Figure 11.1).
The biocontrol agents must not onlybe produced in high yield but also
shouldhavehigh retention of cell viability withmaintenance of crop
compatibilityandbioefficacyduringseveralmonthsof storage. Beyond
production, a satisfactory formulation is necessary to keepthe living agent stable
and infectious 'onthe shelf, which has tobeatambienttemperatureto
accommodate distribution system and on farm storage conditions. An important
feature for successfulcommercialization is the ability todevisearapidand
reliable quality controlprocedure that allowsprecisequantificationof active
ingredient. The biocontrol agent should also provide long-term stability under
warehouse conditions, be compatible with chemical fungicides and insecticides
applied to seedandbecompatiblewithcurrentproduction practices. Any
deviation from these general guidelineswill result in failure.
The
biofungicides that havesucceededthrough the stage of
commercialization have had a critical analysis of market needs and potential. It
is envisaged that fungicides entering markets where they havethe best chance of
performingandwhere the market is receptive to usingbiologicalcontrol
methodswouldhave better chance to succeed.The suitable products shall
provide
competitive
performance, effective formulations
and
economic
production. Performance testing of biocontrol agents under representative

Isolation ofadive
strain agalnst specific/ Iof bioassays
non-speafic pests

F l F
Laboratory Ifermentation I F I
e of user-friendly I pq
evaluations

Figure 11.1 Critical steps for commercialization of biofungicides


252 Chapter 11

cropping conditions, perhaps using a standard fungicide treatment as a control,


will be critical to assure efficiency and dependability of new biocontrol agents
andtoconvincegrowers that the new-fangledmethodsareworthadopting
(Sutton, 1995).It is desirable to providedemonstrated financial return to
producers

3.1 BiofungicideCommercialization - CaseStudies

An insight into various attributes and requisites for successful


commercializationcan also beobtainedfrom the case studies ofvarious
biofungicides. Harman (1996) cited his experience on commercialization of a
patentedbiofungicide Trichoderma harzianum strain 1295-22(Bio-Trek22G)
which was demonstrated to be highly effective seed protectant. He observed that
legal andcommercialconsiderationswereextremelyimportant. Particularly
protection of intellectual property was essential, as without it, few companies
will be interested in developing it further. Besides, adequate business, legal and
financial expertise, availability of large-scale production techniques
and
facilities are also essential. Hofstein and
Chapple(1999)described the
commercializationof Ampelomycesquisqualis (AQ10)for the controlof
powdery mildews (PMD) on grape vines. They found that AQlO did not give
acceptable level of control, once the PMD reaches a certain level of infestation
(> 30%). The product, therefore, has been presented as a preventive treatment
for PMD, if appliedwhen visible incidence is nearzero.Basedonthe
experienceofdeveloping A. quisquafis into commercial
a product,they
concluded that acandidatebiofungicideshouldbethrown into anauthentic
commercial situation atan early stageofdevelopmentto learn about its
attributes. Also, that a biofungicide can not combat devastating fungal pathogens
causing disease such as PMD as a stand-alone treatment and must be offered as
acomponentwithinIPMsystems.BrannenandKenney(1997)have also
attributed the success of Bacillus subtilis strain G B 0 3 (Kodiak) in the cotton
market to its integration with chemical fungicides. Though biological control
agents may havethe possibility of replacing chemical fungicidesin future, in the
near-term use of biologicals require integration with chemical fungicides.

4 Commercialization of Bioherbicides
The whole function of biological weed control is to push the disease process by
tipping ecological balance in favor of the pathogens. This can be achieved by
applying the pathogen in aviable form, atinoculum levels highenoughto
initiate an infection and by manipulating the micro-environment for a long
Commercialization of Biopesticides 2 53

enough period of time to make sure that an infection gets to the point that may
perpetuate differences. However, an approachdirectedatfindingaunique
pathogen that have all the inherent properties necessaryto support a commercial
product may not be successful. On the other hand, developing technology that
can modify the micro-environment and/or enhances the survival of organisms
selected to function as abiocontrolagent in amarket suited forbiological
organism may give more dividends.
Fungal pathogens require some periods of dew on the surface of the
plant for the sporestogerminateand infect the plant. Thedewperiod
requirements may vary for at least 8 hours to as much as 72 hours. As natural
environmentdonotprovidemorethanafewhoursofdew,agreatdealof
inconsistency is found inthe field trial of the fungi. Thusdevelopmentof
technology to the point that it liaise propermanipulation of the micro-
environment, would be a critical requirement for successfil commercialization.
Most applications of bioherbicides in the field provided little or no help to the
organism in serving agricultural function, they are being expectedto provide.
Logical bioherbicide tracks are normally considered to be weeds which
escapechemical controls, weedswhichescape cultural controls, weeds in
environmentally sensitive settings andweeds in organiccroppingsystems.
Ideally a proper track weed would be an agricultural system that would permit
some environmental modifications associated with an economic incentive. Turf,
for example, is generallylocated in environmentally sensitive locationsand
irrigation is part of management system. Thus moving a pathogeninto the weed
wouldnot be a problem.On the otherhand,Charudattan(1988)noted that
College@ wasan attractive candidate for developmentbecausethechemical
herbicides used against northern jointvetch were bannedin the market place for
the herbicides.
CrossandPolonenko(1996) cited animportantlessonfrom the
commercialization experience of BioMala. According to them for commercial
success of biocontrol organisms, a change of focus from product-driven research
anddevelopmenttomarket-drivenresearchandproductdevelopment is
necessary. This requires accurateforecast of customer demandfor the biocontrol
product, total market potential and determination of the needs and benefits that
the product will satisfy.
Bioherbicides also should be
formulated
for
pathogen stability,
persistence, efficacyand application. Inmost cases, shelf life of at least 18
months is critical to successful commercialization. However, chlamydospores of
Phytophthora palmivora formulated as Devine@by Abbott rapidly lost viability
in storage. Therefore, the product was produced anddistributed only after orders
were placed (Kenney, 1986). Refrigeration of other formulated fungal herbicides
appears critical to storage stability.
254 Chapter 11

5 Commercialization of Bioinsecticides
In order to be successful commercially, the bioinsecticides have to directly and
effectively compete with
chemical insecticides in the world market.
Bioinsecticides surpass the performance of chemical insecticides only when the
target pest is resistant to the chemicalbutsusceptibleto bioinsecticide. For
example, Colorado potato beetle is resistant to a variety of chemicals but it can
be effectively controlledby Bacillusthuringiensis. The activity ofsome
traditional bioinsecticides has been enhanced using modified formulations but
this has not resultedin increasing the spectrum of pest control level
or of activity
to match the activity of chemical insecticides. Essentially, significant advances
in activity, persistence and delivery of bioinsecticideswill be necessary to equal
their performance to chemical insecticides.
Inanalysing scientific andbusinessconsiderations to developa
pesticide discovery, it has been found necessary that the product be patentable,
in addition to beingcompetitiveandhaving a widerangeofpestcontrol
(Stowell, 1993). To cite example, Autographa californica nuclear polyhedrosis
virus (AcNPV) has been found to provide effective biological control for many
caterpillar pests similar to Bt bioinsecticides. It has also been proven safe in
extensive toxicology tests and has been demonstrated to be effective in field
performance evaluations. Unfortunately,AcNPVwasnotpatentedafter its
discovery.Thelackof intellectual propertyprotectionand no competitive
advantage as compared to otherbiopesticidespreventeddevelopment of this
technology.
Many major companies in the United States are involved in developing
bioinsecticides. Most agrochemical companies are mainly working on Bt. This
technology is well established and it does not require a high investment. Other
companies are involved in nematode, virus andfungustechnologies. The
progress in mass production
throughfermentation(nematodes, Bt) and
generations in vivo (baculoviruses)wereinstrumental in marketingthese
products. Research efforts are in progress to develop and optimize the in vitro
production process for baculoviruses and fungi.

5.1 Bacillus thuringiensis

Bacillus thuringiensis is the most widely used biological insecticide. The Bt


technology has been developed to the point that its capable of protecting crops
from insect damage. There are persuasive arguments that Bt endotoxins should
be regarded as chemicals. Bt has evolved to produce large quantities of crystal
proteins, making it a logical host for developing improved Cry bioinsecticides.
It is now accepted that engineered forms of the Cry proteins showing improved
potencyor yield, regardlessof their host, makeCrybioinsecticidesamore
Commercialization of Biopesticides 255

attractive and practical alternative to synthetic chemical control agents.


However, the introduction of Bt transgenic plants will have an impact on the
market share ofBr sprays, especially in the cotton market.
Farmers with high hopes of controlling destructive insect pests have
greeted the new Bt products,both plants and sprays, withenthusiasm.The
market is likely to growrapidly as anincreasingnumberofgenetically
engineered pesticides reach the marketandpressuremountstoreplacemore
traditional toxic chemicals.
A comparative economic analysis of a Bt-based product and standard
chemical products has clearly brought out that in specific situations, even non-
engineered Bt products perform comparable to or better than chemical standard
approach. In a 2-year long study on fresh-market tomato plantings employing
separately a Bt-product Javelin and methomyl and permethrin, net profits were
found to be equal to or better for Bt product application than chemical standard
approach.Otherlong-term benefits includedreducedfrequency of chemical
application and, negligibleeffect on natural enemies (Trumble et al. 1994).

5.2 Baculoviruses

It has been believed that recombinant baculovirus research would result in a


unique,competitive insecticide that wouldcontroleconomicallyimportant
insects withno effect on beneficial species. It couldbesafelyappliedto
vegetables up to harvest, demonstrate no adverse ecological effects and present
no toxicological hazards to mammals. DuPont and American Cyanamid, both
have recently tested recombinant nucleopolyhedro viruses (NPVs) in selected
cotton and vegetable growing areasof the United States against cabbage looper,
cotton bollworm, tobacco budworm, beet armyworm, and diamondback moth. In
anticipation of the wide-scale use ofrecombinantNPVs for the control of
lepidopterous pests, effective control strategies are being formulated and tested
to ensure the success and acceptance of this new technology. DuPont is taking
its new scorpion-loaded virus on the road. This material combines a scorpion
venomgenewithabaculovirus Autographa californica (McCutchenand
Flexner, 1999).
It is stipulated that a genetically engineered virus would need to achieve
sales of$40million in ordertojustifyanR&Dexpenditureof$15million
required. Zeneca,a British life sciencescompany,terminatedaproject on
recombinant baculoviruses as they believed that many issues remained that must
be grappled with and solved before sufficient balance is achieved between the
efficacy the growers are happy with and apractical ability to make biopesticides
available to thesegrowers(Broadhurst,1998).Thebusinessobjectivesof
Zeneca for genetically engineered baculoviruses were outlinedas follows:
Chapter 1I

The genetically engineered baculoviruses to have an adequate effect on


lepidopteran pests in cotton, vegetables and other crops suchas corn.
The achievable effect is to be equivalent to chemical standards on the
least effective end of the spectrum for control oftarget pests, e.g. thiocarb
in cotton.
A method to produce sufficient quantities of the engineered virus with
consistent quality and cost effectively. This requirement was believed to
stipulate vitro
in production
methodology
with
manyassociated
challenges. For example, use of insect cell cultures on large scale under
sterile conditions, chiefly driven by an assumption that traditional in vivo
methodswouldnotbeapplicable when the larvaediedmuchmore
quickly.
Robustformulationtechnologyassociatedwith the formidabletaskof
working with biologicalmaterials.

The recombinant baculovirus products have yet to be commercialized


butan insight into their deploymentscenarios is available. Thesecouldbe
utilized for controloflepidopteranpestspecies as biocontrolagents in
rotational-use strategies, to relax selection pressureexertedbysynthetic
insecticides or evenby Bt in pestpopulations, for use in combinationwith
certain chemical insecticides; and as augmentation of transgenic crop varieties
andestablishment of binarytransgenicplantresistancehiocontrolsystems
(Treacy, 1999).

5.3 Fungal Bioinsecticides

Fungi are potential bioinsecticides. Their use is a commercial reality and there
are a number of fimgi suchas Beauveria and Metarhyzium that are widely used,
especially in greenhouses.MycotechCorporationhasregisteredseveral
formulation of B. bassiana strain GHA (BotaniGard@) targeting the silverleaf
whitefly, thrips and aphids. As silverleaf whitefly inhabit primarilylower
surface of plant leaves, maximum coverage of leaf undersides is obtained by
spraying upward from below canopy level. For commercialization, the critical
need for intensively replicated field experimentsand large scale trials under
commercial production
conditions
can
not
be overstated. Technological
breakthroughs will also be needed, especially in production and formulation, to
extend their uses to large acreage areas (Georgis, 1996). Additional research is
needed especially with the aim of improving product efficacy and consistency
under variable field conditions.
Commercialization of Biopesticides 257

6 Commercialization of Pest-Resistant Crops


The expression of Bt genes in plants has provided a widespread application of
genetic engineering byshifting the paradigm of external spraying of pest control
agents to internalizing this activity within the plant. This approach has provided
advantage of effective andenvironmentallyacceptablepestcontrol in large-
acreage crops such as cotton and corn and eliminated or reduced the need of
repeated foliar application of pest control agents.

6.1 Insect-ResistantCotton - A Case Study

An account of Monsanto's journey through development of BollgardB cotton


from laboratory to market place, spanning a nearly decade of efforts, has been
provided by Perlak and Fischhoff(1996), Sims (1996) and Goldberg and Tasker
(1997) (Figure 11.2).

6.1.1 Insect-Resistant Plants - StrategicPositioning

Based on successful research in introducing and expressing the protein-toxin


genes in tobacco and tomato plants and finding them insecticidal in laboratory
andgreenhousebioassays, the identification ofakeycrop for the first
introduction of insect-resistant plants was considered a necessity by Monsanto.
The factors in choosing cotton as a focus crop included technical feasibility of
transformation, high value crop to justifythe effort and consumer acceptance of
the technology. It is a dicotyledonous plant
and
hasbeen
successfully
regenerated and transformed by Agrobacterium-mediated transformation. Cotton
wasoneof the mostchemical-intensivecropsgrown in approximately 12.5
millionacres in the United States alone. Despiteheavychemical use, on
average, farmers still lost up to 20% of their crop to insect pests. Insect damage
remained a serious problem with the b o l l w o d u d w o r m complex consisting of
tobacco budworm (Heliothis virescens), cotton budworm (Helicoverpa zea) and
pinkbollworm(Pectinophora gossypiella). These are all sensitive to Bt
endotoxins.

6.1.2 TechnicalAdvancesRequired

Whiletechnical feasibility ofcottonregenerationandtransformationwas


known, its efficiency was low. A commitment to efficient plant transformation
was required followed by screening of close to 1000 transgenic cotton plantsin
Chapter 11

__c Technical feasibility of transformation


Identification of
__C High value crop

Acceptance of technology

n
__c Enhanced efficacy of transformation
Technical advances

Increased level of gene expression

__C Protection from pest damage and yield


Field testing
Field performance comparableto
chemlcal pestiude treatment

-- Food and feed safety


Effect onnon-target organisms
Weediness potentialof transgenics
Pest resistance development potential
Product characterization induding
host range and selectivlty
Environmental and digestive fate
studies ofprotein
Homology and allergeniaty
Ecological and environmentalrisks
Possibility of gene transfer
+ Methods for abatement of resistance
development

relationship of &-biotech/
seed company Cotton plant genepool of the seed
__c ampanv to provide a vehicle for
Ag-biotechs.capabilities

Figure 11.2 Stages of laboratory to commercialization of Bt-cotton


Commercialization of Biopesticides 259

a green house test with cotton bollworm for identification of best plants for
back-crossingprograms into commercial "elite" lines. Newapproacheswere
initiated for increasedexpressionof crylAb proteingenes in genetically
modified cotton. Site-directed mutagenesis was employed topartially modify the
gene by removing the DNA sequences predicted to inhibit efficient expression.
This gene increased the expression in plants by 100-fold compared to the wild-
type (WT) gene. Another approach used a fully modified synthetic gene that
encoded a protein identical in amino acid sequence to the WT gene. Consequent
to modification of DNA sequence, expression levels in plants increased up to
1000-foldover levels seenwithwild-typegenes in plants. This directly
correlated to increased insecticidal activity. Theincreasedexpressionwas
generic across several plant species including tobacco, tomato, cotton and corn
andwasobtainedwith two distinct genes, c l y l A b andamodifiedtruncated
crylAc gene. The modified genes were introduced in cotton resulting in very
high levels of insect-control protein gene expression.

6.1.3 Field-testing of Insect-ResistantCotton

The improvedtransformationsystemand the increased levels of target gene


expression
were
combined to generate
plants suitable for field-testing.
Beginning from the day a cotton transformation wasinitiated to the day the first
preliminary results are obtained from the field, it took over two and half years.
Theplantsdemonstratedexcellentprotectionfromlepidopterous pests. The
protectionobtained was foundcomparableto that obtainedusingweekly
treatment of chemical insecticides for lepidopteran control on non-transformed
plants. The yield of cotton from transgenic cotton in the field tests was also
comparable to the insecticide-treated non-trangenic cotton.

6. I .4 RegulatoryIssues

The Bt proteins are generally regarded safe; the issues related to food and food
safety, effects on non-target organisms, weediness potential of transgenics and
also the potential for pest resistance developmentwereinvestigated(Sims,
1996). Besides, when one deals with microprojectile bombardment of a cell for
constructing a recombinant DNA system,it is difficult to get a single copy of the
gene go into the plant cell cleanly. Often, either multiple copies or partial copies
result (see figure 3.1) and these were required to be defined and described to the
regulatoryagency,EPA.Theproductcharacterizationincludedhostrange
studies and verification that the proteins are indeed selective to target pest
species. A homology search was carried out and allergenicity was determined to
find if the Bt protein is like any known toxins or allergens. Digestive fate studies
260 Chapter 11

were performed in vitro using gastric- and intestinal-fluids at different pHs to be


sure that protein digested.
Ecological and environmental risks were evaluated for adverse effects
on non-targetorganisms: avian, mammalian, aquatic, beneficial insects and
endangered species. These tests were unique to the crop and pests involved. For
example, for Bt-corn, tests wereconductedon the potential effects onfish
because field cornmay be manufactured into commercial fish food. Also
investigated were the issues related to genes such as possibility of their transfer
to other plants or problems caused in the plant itself. Environmental fate of the
protein was assessed to see if it accumulated and became a problem to non-
targets, especially in the soil, and to honeybee, ladybeetle, hymenoptera
(parasitoids) and lacewing. Methods for abatement of resistance development
were also investigated.

6.1.5 Pooling Technologies and Resources for Cotnnzercial Value Sharing

Monsanto needed a seed companyto provide the vehicle for taking their genetic
engineering capability to market. Delta and Pine Land (D&PL), a Mississippi-
based cotton seed marketleader, stood to have an industry advantage if it could
be the first and possibly the only company to market a transgenic cotton product
that worked.D&PLmanagedoneof the largest cottonplantgenepooland
needed to capture some value for the use of its germplasm as a transport agent
for Monsanto's biotechnology. It had a reputation for consistently providing the
highest-qualitycottonseed varieties that gave the farmer the right yield,
optimum maturation cycle andfiber quality characteristics.
However, there was an uncertainty of the value of Bt cotton seeds use
that would be created for those involved in the value chain. These included the
farmers, the seed companies, the technology provider, the distributors and the
agrochemicals companies who stood to loose money through decreases in sales.
It was clear that there would be a complete restructuring of the industry before
the allocation of that value was h l l y understood.
MonsantoandD&PLdrew the first contract in 1992, that was
subsequentlyamended in 1994and1996.TheFebruary1996revisionmade
provision for Monsanto to license its technology directly to the farmer and the
retailer being required to attend a training session and to sign a contract. The
goal was to communicate the value of newly purchased seed with biotech traits
and dissuade the farmer from saving his Bt cottonseed with a high penalty if
found guilty of savingit.
The direct licensing agreements as a marketing process seemed to be
phenomenalsuccessandaddedconsiderablevalue for bothMonsantoand
D&PL. This generated an extensive database of the cotton farmers' businesses
through the grower licensing agreements and established a precedent of direct
Commercialization of Biopesticides 26 1

marketing to the consumer in the agriculture industry. It provided an openingfor


Monsanto, in the future, and with other biotech products, an ability to implement
a strategy ofmasscustomization,marketing directly tofarmer,constructing
individual deals (by product and geography) that optimized value. On the other
hand, from the seed company perspective, they made more money per acre from
the transgenic cotton fee than on the non-transgenics seed margin. They could
also demonstrate the 'leap of faith required by farmers in the product they sold
based on their reputation of quality seed company'.
Lessonsfrom the commercializationoftransgeniccottonwould
predict that anticipatedcomplexitiesof restructuring mustincludeproviding
patent protection, a system for collection of royalties and changes in current
farm and industry structures, including partnerships that incorporate access to
enablingtechnologies into traditional seedstockcompanies(Goldbergand
Tasker, 1997).
Similar to Monsanto joining hands with D&PL for the development of
Bt cotton lines, Mycogen who developed new corn hybridsresistant to European
corn borer, found that it had neither the time nor the resources to go it alone in
developingnewengineeredplant varieties andsoughtpartnership to pool its
technologyand resources. MycogencollaboratedwithPioneerHybrid for
developmentof insect resistant cropsbased on Bt. Suchcollaborationby
combining complementary areas ofexpertise are expected to allow companies to
make rapid progressin development of newagricultural products.

7 Concluding Remarks
There are a number of critical factors that contribute equally to the ultimate
success of a product development and commercialization program of biologicals
(Leggett and Gleddie, 1995). All of these are broadly in conformity with the
experiencegainedfromalreadycommercializedbiopesticidesandcan be
summarized as follows:
1) Sufficient customerdemandandmarket size to ensurea financial return
on the investment in researchanddevelopment in areasonabletime
period;
2) a cost-effective manufacturingprocess;
3) stable, effective andeasy-to-useformulations;
4) productcompatibilitywith existing distribution systemsand agricultural
practices;
5) highly efficacious, reliable andreproducible field performanceby the
formulated product;
6) the ability to obtain patent protection and product registrations and
7) sufficient salesrevenuesgenerated to supportcontinuedmarketingand
promotion, and to continuefurther product developments.
262 Chapter 11

Contact chemical agents have conditioned growers to evaluate control


based on quick knock down. There are few sights more gratifying to a grower
than that of yield-threateningpests perishing shortly after a chemical pesticideis
applied. Biologicals work more slowly, requiring different, more complicated
criteria such as yield for measuring effectiveness. Therefore,achange of
mindset is also essential for encouragingbroader use of biopesticides. This
transition will requiregrowerstobe better educatedaboutbiologicalcontrol
technologiesthroughcooperativeextension services. For this to happen,
extension personnel who have immense knowledge base to support the use of
chemical pesticides will have to be provided with new knowledge for biological
agents for transmittal to growers.
Commercialization of genetically engineered crops that began in 1996
withready
a acceptancebyfarmers attained animpressiveandrapidly
expandinggrowth.Farmersaccepted Bt corn, for example,much faster as
compared to hybrid corn. However after four years of explosive growth, by late
1999,increasingconsumerconcernstowardsenvironmentalimpactandfood
safety had resulted in a backlash. Environmental issues related to biodiversity,
gene pollution, gene transfer, and insect resistance and food safety issue related
to antibiotic resistance, allergic reactionsandhealth effects, hadcreated
controversiesand distrust. Manyofthese issues remainunresolved, as no
unequivocalanswerswere available yet. If perceptions create reality in the
market place, then reports such as transgenicpotatoesfed to rats causing
thickening of the walls of part of their digestive tract (Ewen and Pusztai, 1999)
and laboratory mortality of monarchbutterfly larvae fed on pollen from Bt corn
(Losey et al., 1999), have not helped the matters. This may have an effect of
stunting the growth of genetically modified crops market, at least temporarily.
By the end of 1999,Monsantohadagreedtomodify their genetechnology
licensing agreements with Delta & Pine Land for insect-resistant and herbicide-
tolerant crops and abandoned the plans of its acquisition, setting it free to talk
to other potential suitors. Subsequently, Monsanto has merged with Pharmacia
& Upjohn with a speculation of its agribusiness would be spun off in a public
offering.
Themarketappears tobe drivenbyperceptions that determine
consumerchoice rather than rationale in termsof the safetyalone.The
agronomic or input traits designed withan eye to farming practices and
pesticide use are visible only to farmers. Positive environmental effects, such as
decreased pesticide use are seen beneficial but the consumer perceives real value
in termsoflower prices andor better food quality. Someof the surveys,
however, give hope that acceptance will grow as consumer knowledge about
biotechnologyincreases(Thayer,1999). Particularly, advancementstowards
next-generation genetically modified crops with improved output traits, such
as tangible benefits for health and nutrition might even create demand and be
crucial in winning over the consumers. In the end, consumers are the ones who
Commercialization of Biopesticides 263

will make the choices and will ultimately drive the market, not technology. But,
more and better science-based information must be made available to help them
with these choices.

References

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controlproduct for suppressionofsoil-borne plant pathogens of cotton. J.
Ind. Microbiol. Biotechnol.. 19. I69 - 17 I .
2. Broadhurst, M.D., 1998.Biopesticides: From disillusionmentto integrated
cropmanagement- A large company perspective. Abstracts, Alternative
Paradigms for Commercializing Biological
Control
Workshop. Rutgers
(Jniversity,
New Brunswick.
NJ, Internet: http://www.rci.rutgers
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http://www.nysaes.cornelI.edu/ent/bcconf/talks/georgishtml.
10. Goldberg, R. A. and C. Taskcr. 1997. Delta & Pine Land: Measuring the value
of transgenic cotton. I-larvardBusiness
School Case Studv 9-597-005
(Revised). Harvard Business School Publishing. Boston.
11. Harman, G. E.. 1996. Trichoderma forbiocontrol of plant pathogens: From
basic research to commercialized products. Confirence on Biological Control,
Cornell IJniv..April 11-13. 1996. Internet: I ~ I I ~ : / / N u u . I ~ sacs.cor.ncll.cdu/c.n(/
hcconfitalks/l~arman.html.
12. Hofstein, R. and A. Chapple. 1999. Commercial development of fungicides.
in Biopesticides: Use andDelivery, F.R.Halland J.J. Menn, eds.. tlumana
Press, Totowa, NJ, pp.77-102.
13. Kenney, D. S.. 1986. Devine@- The way it was developed - an industrialists
view. Weed Sci.. 34. 15-1 6.
Chapter 11

14. Legget, M. E. and S. C. Gleddie, 1995. Developing biofertilizer and biocontrol


agents that meet farmers expectations,Adv. Plant Pathol., 11,59-74.
15. Losey, J. E., L. S. Rayorand M.E. Carter,1999.Transgenicpollenharms
monarch larvae, Nature, 399, 214.
16. McCutchen, W. F. and L. Flexner, 1999. Joint actions of baculoviruses and
othercontrolagents, in Biopesicides:Use and Delivery, F.R.HallandJ.J.
Menn, editors, Humana Press, Totowa, N.J., pp. 341-355.
17. Perlak, F. J. and D. A. Fischhoff,1993. Insect-resistantcotton:From
laboratory to the marketplace, Advanced Engitteered Pesticides, L. Kim, ed.,
Marcel Dekker, New York, NY, pp 199-21I .
18. Shimoda, S.M. 1997. Challenges of commercializing biopesticides in a more
competitivemarketplace, Second Annual Conferewe on Biopesticides and
Transgenic Plants: New Technologies to Improve Eflcacy, Safety and
Profitability, Washinton D.C., Jan. 27-28, 1997.
19. Sims, S., 1996.Developmentandcommercializationofinsectresistant
transgenic crops, Conference on Biological Control, Cornell Univ., April 11-
13, 1996, http://www.nysaes.cornell.edu/ent/bacconf/talks/sims.html.
20. Stewart, R. S., 1999. Toward one market: harmonising efforts between the US
andCanadamay be threatened by FQPAand CMOS, Farm Chemicals
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21. Stowell, L. J., 1993.Factorsinfluencingacceptanceanddevelopmentof
biopesticides,in Advanced Engirleered Pesticides, L. Kim, ed., Marcel
Dekker, Inc., New York, pp. 249-260.
22. Sutton, J. C.,1995.Evaluationofmicroorganismsforbiocontrol: Botrytis
cinerea and strawberry, a case study,Adv. Plarlt Pathol., 2, 173-190.
23. Thayer, A. M.,1999.Agbiotechfood:risky or riskfree,Chem. & Engg.
News, 77(44), 1 1-20.
24. Treacy, M. F., 1999. Recombinant baculoviruses, in Biopesticides: Use and
Delivery, F. R. Hall and J. J. Menn, eds., Humana Press, Totowa, NJ, pp. 321-
340.
25. Trumble, J. T., W. G. Carson and K. K. White, 1994. Economic analysis of a
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26. Wilson,C. L. and M. A.Jackson,1997.Commercializing of biologically-
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19, 156.
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Glossary, and
Product, Manufacturer,
Pathogen and Subject indices
This Page Intentionally Left Blank
Glossary

Actinomycete Filamentous bacteria that have somctinles been classified as a fungl


(Literally imperfecti.Thcname is often used to rcfer spccificallytothose
"ray fungi") species that form mycelium.

Adjuvant Material added to improvc some chemicalor physical propcrty (c.g.,


of a plant protectant) such as solvents, diluents, carrlers, cmulsificrs,
stickers orspreaders.

Agroecosystem A relatively artificial ecosystem In an agricultural field, pasture, or


orchard.

Allele Any of onc or more altcmative forms of a givcn gene;both (or all)
alleles of a given gene arc concerned
with the same trait or
characteristic.

Antagonism An ccologlcal association bchveen organisms in whlch one or more


of thc participants is harmcd or has its actlvltles limitcd.

Antagonist An agent or substance that counteracts the action


of another.

Antibiosis An associatlon between two organisms in whlch one harms the


other.

Apothecium An ascus-bearing structure (ascocarp) in which the ascus-producing


layer (hymenium) is not coveredby fungal tissue at maturity.

Appressorium An enlargement on a hypha or germ tube that attachcsitself to the


host before penetratlon takes placc.(PI. appressoria.)

Arthropod A phylum or division of the animal kingdom: include insects;


spiders and crustace.

Autosome All chromosorncs except the sex chromosomes.

Bacteriophage Virus that livesin and kills bacteria. Also called phage.

Biological The deliberate use by humans of one species of organism to


control climinate or control another.

Biotroph An organism that derives nutrients from the living tissuesof another
organlsm (its host).
Blastospore A spore that arlses by budding, as
in yeasts.

267
Glossary

Brush border A superficial protoplastic modificationIn the form of filiform


membrane processes or microvilli present on certain absorptic cellsin the
ntestinal epithelium and the proximal convolutions of nephrons.

Chlamydospore A loosely used term usually applied to athick walled, asexually-


produced resting spore formed by certain fungi; the termis
sometime used to refer specifically to the teliospore of the smut
fungi.

Cloning An i n vitro procedure In which a particular sequence of DNA(e.g. a


gene) is reproduced in large amountby inserting (splicing) it into a
suitable replicon (the vector or cloning vector), introducing the
resultant recombinant (hybrld) molecule into acell In which it can
replicate and finally growing the cellsin culture.

Columnar Epithelium distinguished by elongated, columnar or prismaticcells.


epithelium
Cytolytic Able to lyse cells.

Damping-off Collapse and death of seedling plants resulting from the


development of a stem lesionat soil level.

Dicotyledon Plantwhoseseedshavetwocotyledons or seedleaves,suchas


(Dlcot) beans.

Deuteromycetes A large miscellaneous artificial group of fungiin which sexual


reproduction does not occur or has not been found, contains most of
the wilts and some damping off fungi.

Endogenous Growing throughout the substance of the stem, instead of by


superficial layers.

Endoparasite Any of the various parasites which lives within the body its
ofhosts.

Endophyte An organism parasitic partlyor wholly within a plant.

Endospore A type of spore formed intracellularly by the parent


cell or hypa and
are formed under conditionsof nutrient limitation.

Endotoxin A toxin produced withln an organism and liberatedonly when the


organisms disintegratesor destroyed.

Entomogenous Growing in or on insects.

Entomopatho- Insect-attacking organism.


genic
Glossary 269

Entomophagous Anyinsectthateatsotherinsects.
insect
Entomophagous An insect or fungus that parasites insects.
parasite
Enzyme A large
complex protein
molecule
producedbythe
body
that
stimulates or speeds up various chemical reactions without being
used up itself; an organic catalyst.

Epiphyte A plantgrowingonanother(usuallynot fed byit) or amicro-


organism living on the surface of plants in a non-parasitic
relationship.

Epithelium A primary animal tissue, distinguished by cells being close together


with little intercellular substance covers free surfaces and lines body
cavities and ducts.

Epizootic A disease outbreak within an insect population.

Erythrocyte A type of bloodcell that contains a nucleus inall vertebrates. Also


known an red blood cell.

Exogenous 1) Produced on the outside of another body, 2) produced externally,


as spores on the tipsof hyphae, 3) Growing by outer additionals of
annual layers, as the wood in dicotyledons.

Exospore or by
A type of spore formed form the parent organism by budding
septum formulation and fission.

Exotoxin A soluble toxin excreted by specific bacteria and absorbed into the
tissues of the host.

Expression In genetics, manifestation of a characteristic that is specified by a


gene. With hereditary diseases, for example, a person can carry the
gene for the disease but not actually has the disease.In this case, the
gene is present but not expressed. In industrial biotechnology, the
term is often used to mean the production of a protein by a gene that
has been inserted into a new host organism.

Facultative Designating an organism which is capable of living under more than


one condition: e.g. a saprophyte and as a parasite; asan aerobic or
anaerobic organlsms.

Genetic A process that resultsin a change In the genetic make up of a


modification population. Methods of inducing genetic modification include
conjugation, in vivo rearrangements of transposable elements,in
vifro gene recombination techniques, protoplast fusion, the use of
mutagens, hybridization by normal breeding techniques.
270 Glossary

Genome The total hereditary


material of cell,
a comprlsingtheentire
chromosomal set (hence of genes) foundin each nucleus of a given
specles.

Genotype The
genetic
constitution
an
organism.
of

Goblet cell Aunicellular,mucus-secretingintraepithelialglandthat is distended


on the free surface.

Gram stain Astainingmethoddevised by DanishphysicianHansGrams to aid


in the identification of bacteria. Bacterla either resist discolorization
with alcohol and retain the initial deep violet stain (Gram positive)
or can be decolorized by alcohol and are stained with a contrast stam
(Gram negative).

Hemocoel An expandedportionofthebloodsystem in arthropodsthatreplaces


a portion of the coelom.

Hemolymph Thecirculatingfluidoftheopencirculatorysystemsofmany
invertebrates.

Hemolysis Thedestructionof red bloodcellsandresultingescapeof


hemoglobin.

Heterozygous An anlmalthatcarriesgenesfromtwodifferentcharacters(Impure).

Homologous Occuronlybetweentwosequencesthathavefairlyextenslveregions
recombination homology.
of

Hyperparasite An organismthat is parasitic on anotherparasite.

Hypha Thesimple or branched


thread
like
filaments
that
compose
the
web
like mycelium of fungi.

Inoculum Thatportionofindividualpathogen or its partsthatcancause


infection and that is introduced into or transferred to a host or
medium.

Intraoccular Withintheglobeoftheeye.

Intraperitoneal Within the cavity of the body that contain the stomach and
intestines.
In vitro Frequently in thesensenotundernaturalconditions e.g. in the
(literally in glass) laboratory; in experimental culture (of chemical reactions) not in
living cells.

I n vivo Inliving
organism.
the
Glossary 27 1

Lesion localized
A areadiscolored,
of diseased
tissue.

Lethal The
lethal
concentration
(written
as LClo or LCso or LClooor any
concentration percentage)median is thepartspermillion(ppm) or parts(LC)per
billion (ppb) of toxlcant in water or air which kills I O or 50 or 100%
respectively of the target speciesin a 24-hour perlod.

Lethal dose The


lethal
dose
(written LDlo, LDSO,LDloo or any
percentage)
(LD) median is themilligrams
toxicant
of perkilogram body
of weight
that kills 10, 50 or 100% of the target species.

Lysis The enzymatic dissolution of all or part of a uni- or multi-cellular


structure or dissolution of a phage-infected bacterium.

Metamorphosis Aprocessbywhich an organismchanges in formandstructure in


the course ofits development, as many Insects do.

Microvillus Oneofthefiliform(threadlike or filamentous)processesthatforma


brushborderonthesurfacesofcertainspecialized cells, suchas
intestinal epithelium.

Mildew A fungaldiseaseofplants in whichthemyceliumandsporesofthe


fungus are seen as a whitlsh growthon the host surface.

Mold Any
profuse or woolly
fungusgrowth
on
damp
decaying
or matter
or on surfaces of plant tlssue. Blue mold or green mold is caused by
Penicillium spp. and grey mold,by Botrytis crnerea.

Monocotyledon Plant having a single cotyledons or seed leaf such as corn.


(Monocot)
Mutant A variant,
different
genetically
andoften
visibly
from
its
parent or
parentsand awing rathersuddenly or abruptly.Of an organism,
population,
gene,
chromosome, etc.: Differing
from
the
corresponding wild typeby changes in one or more loci.

Mutation A sudden random change in the genetlc material of a cell. A stable,


heritable change in the nucleotide sequence of a genetic nucleic acid
(DNA, or RNA in viruses,viroids,etc)typicallyresulting in the
generation of a new allele and a new phenotype. Mutation can occur
naturallyor can beinduced by radiation(X-ray,gammarayor
thermal neutrons) or chemically.

Mycelium Thehypha or massofhyphaethatmakeupthebodyofafungus.A


mass of interwoven filamentous 'threads' that make up the vegetative
part of a fungus. Mycelia(PI.).

Mycoparasite Afungusparasiticonotherfungi.
272 Glossary

Mycosis An infection by a parasitic fungus,or a disease so caused.

Mycotoxins Chemicalsubstancesproduced by fungithatmayresult in illness


or feed containing them
and death of animals and humans when food
is eaten.

Necrotroph An organlsm that derives nutrients from dead plant or animal tissues,
whether or not it is responsible for the deathof those tissues.

Neurotoxicity The state or condition of being poisonous to the brain and nerves of
the body.

Obligate Descriptionof an organlsmthat


requires
particular
a setof
environmentalconditions or nutrlentforgrowth or survival. For
example, an obligateaerobewillonlygrow in thepresence of
oxygen and an obligate anaerobe's growth will be inhibited or will
be killed in the presence of oxygen.

Obligate A parasite that in nature can grow and multiply only on or in living
parasite organisms.

Oospore Athick-walledsporeproducedbysexualreproductionindowny
mildews and related fungi.

Parasite An organism that lives at least for a time on or in at the expense of


living anlmals or plants.

Pathogen Anymicroorganismwhichbydirectinteractionwith(injectionof
another organism (by convention, a multi-cellular organism) cause
disease in that organism.

Pathogenicity The ability of a pathogento cause disease

Peritrophic Achitinoustubefreewithinthecavityofthemidgutofinsects
membrane which separates food from the lining epithelium.
Phenotype Individualsofthesamephenotype look alikebutmaynotbreed
alike.

Phylloplane The surface(s) of aleaf.

Phytopatho- Term applicable to a microorganism that can incite disease


in plants.
genic
Phytotoxic Toxic to plantsor plant growth.

Plasmid A circular piece of DNA found outside the chromosome in bacteria.


Plasmids
are
the
principal
tools
for
Inserting
new
genetic
information into the microorganismsor plants.
Glossary 273

Plasmid vector A plasmld used as cloning vehicles or vectors for the introduction of
foreignDNA-containinggenesthat do notnormallyoccur in the
host cell.

Polyphagous A parasitewhich is capableofparasitizingconsiderablenumberof


parasites species.
host

Propagule Thepartof an organismthatmay be disseminatedandreproducethe


organism.

Proteinase An enzyme that


digests
proteins
and
acts
directly
on
the
native
proteins in their conversion to simpler substances.

Proteolytic Anyenzymethatcatalyzesthebreakdownofprotein.
Enzyme
Pycnidium Aclosedsporocarp,usuallyopening by apore,thatcontainsacavity
bearing conidia.

Recombinant Thetechniqueofisolatingageneandinserting it intothe DNA of


DNA technology anotherorganism,alsocalledgeneticengineering,genesplicing or
genetic modification.

Restriction Enzymesthatcan be usedasmolecularscissorstocutDNAinto


Enzymes reproducible
fragments
specific
at sites. Thereare
many
different
kinds of restriction enzymes and a large number of them have been
carefully catalogued according to the point at which they will cut a
DNA molecule. By selecting the appropriate restriction enzyme, a
given DNA molecule canbe cut at a useful site.

Rhizosphere Thesoilsregion on andaroundplantroots.Therootzone,thearea


where microorganisms are most active in increasing the availability
of nutrients for plants.

Rot The
softening,
discoloration,
and
often
disintegration
succulent
aof
or bacterial infection.
plant tissue as a result of fungal

Rust Anyof
various
plant
diseases
causmg
to
form
rust-colored
spores on
affected plants.

Saprophyte Plants,
including
certain
bacteria and molds,
capable
of
obtaining
nutrients and energy from dead organic matter.

Sclerotium Hard,
resistant,
multicellular
resting
body,
that
under
favorable
conditions can germinate to produce mycelium or sexual or asexual
fruiting bodies. Sclerotia (PI.).

Senescence The
process
growing
of old.Decline or degeneration,
with
as
maturation, age, or disease stress.
274 Glossary

Septicemia Blood
poisoning,
disease
a condition
which
results
from
the
presence of toxinsor poisons of microorganisms in the blood.

Serotyping Strains are distinguished on the basis of difference in their surface


antigen.

Siderophore A metabolic product of a fungus (or other organism) which binds


ironandfacilitates its transportfromtheenvironmentinto its
microbial cell.

Site-directed The process of introducing specific base-pair mutations into a gene.


mutagenesis

Smut Any of a number of plant diseases characterized by masses of dark,


powdery,andsometimesodorousspores(e.g.,stinkingsmutof
wheat, common smut of maize).

Species A unit used in the classification of living organisms. Members of the


same species resemble each other closely. In the naming of plant
andanimalsLatin is used. Each kindofplant or animalcan be
identified by genus (Plural: genera) and species (both singular and
plural) eg. generic name (genus) of corn1s Zea and the species name
is mays.

Sporangium A fungus fruiting body that produces non-sexual spores within its
external walls.

Spore A discrete sexual or asexual reproductive unit, usually enclosed by a


rigid wall, capable of being disseminated. The reproductive unit of
fungi consisting of one or more cells; it is analogous to the seed of
green plants.

Strain A sub-species group of organisms distinguishable from the rest of


the species by a heritable characteristic that the individuals in the
group have in common.

Stroma Acompactmassofvegetativetissue,sometimesintermixedwith
hosttissue,oftenbearingsporocarpseitherwithin or upon its
surface. (PI. stromata).

Synergism The association of two or more organisms acting at one time and
effectingachangewhichoneonly is notabletomake.The
concurrentparasitismofhostbytwopathogens or effect ofa
chemical in which the symptoms or other effects produced are of
greater magnitude than the sum of the effects of each pathogen or a
chemical acting alone.

Subcutaneous Situated or occurring beneath the skin.


Glossary 275

Toxinomy Division(inbotanicalschemes) or phylum(inzoolyicalscheme),


heirarchy class; order; family, genus, and species.

Transgenic An organismthathasbeentransformedwithforeign
a DNA
sequence.

Transgenic Bt A gene from the Bf bacterium is inserted into the DNA of a plant
plants cell. When the cell is re-generated or grown into a whole plant, the
resultant plant, is resistant to specific insect pests.

Ubiquitous Occurrmg everywhere as houseflies, weeds.

Virion The infectious unitof a virus.

Virulent Capable of causing a severe disease; strongly pathogenic.

Wilt A plant disease characterized by loss of turgidity, which results in


drooping of leaves, stems, and flowers.

Zoospore An asexual, motile reproductive cell, which, by repeated divisionsof


the protoplast, develops into a new plant: a means of reproductionof
many green algae and lower fungi.

Zooplankton Tiny animals which drift with the currents.


This Page Intentionally Left Blank
Product Index
(Product and its manufacturer)

A
Able,79, 79t, 1 1 1 Thermo Trilogy
Agree, IO, 79, 79t, 1 1 1 Thermo Trilogy
AquabacXt,57, 103 Becker Microbial Products
AQ'O, 183t, 183, 192,252 Ecogen
Aspire, 185t, 186, 192 Ecogen
Attribute, 94t Novartis

Bactimos briquette, 1 12, 238 Summit Chemical


Bactoculicide, 103, 104t Biological Preparation
Binab T, 174t, 179, 179f Bio-Innovation AL
Bio 1020, 2 19, 220t Bayer
BioBlast, 219, 220t EcoScience
BioCoat, 171t, 173 S&G Seeds
BioMal, 213t, 214, 218,253 Philom Bios
BioPath, 2 19, 220t EcoScience
Bio-Savel I , 185, 192 EcoScience
Bio-Save 1 IO, 185t EcoScience
Bio-Save 1000, 185t EcoScience
Bio-Trek 22G, 173, 191 BioWorks
Biotoxibacillin, 8 (Produced in Rusia)
Blight Ban A 506, 183t, 193 Plant Health Technologies
Blue Circle, 171t CCT Corporation
BMP 123,48t Becker Microbials Products
Bollgard, 92t, 137, 138, 257 Monsanto
BotaniGard, 139,256 Mycotech Corporation
Bt-Xtra, 94t DeKalb

C
Caprovirusine, 15 1 t Natural Plant Protection
Casst, 213t, 213,218 Mycogen Corporation

277
278 Product Index

Collego, 213, 213t,214,217,218,220, Pharmacia & Upjohn, Encore


253 Technologies
Condor, IO, 79,79t, 1 13 Ecogen
Conquer, 171t, 172 Mauri Foods
CryMax, SSt, 89, 112 Ecogen
Cutlass, IO, 79,79t, 111 Ecogen
Cyd-X, 15 1t Thermo Trilogy

D
Dagger G, 171t, 172 Ecogen
Delfin, 484 1 13 Thermo Trilogy
Deny, 171t, 173, 185t, 193 CCT Corporation
Design, 11,79, 79t Thermo Trilogy
DeVine, 2 13t,2 14,217,220,253 Abbott (Now Valent Biosciences)
Dibeta, 8 Abbott (Now ValentBiosciences)
DiPel, 44,48t, 52, 108, 1 1 I , 112, 113, 114, Abbott (Now Valent BioSclences)
115, 125,234, 235, 236t
Dr. Biosedge, 2 13 t,2 14 CCT Corporation,
Tifton Innovation Corporation
Duplex, 238

E
Elcar, 151t, 155 Novartis
Epic, 170t, 172 Gustafson
ESC 170 GH,220t EcoScience
Exobac, 8 Caffaro

F
Florbac FC, 11 1 Novo Nordisk
Foil, I O , 79, 79t Ecogen
Foray 488, 1 14t Novo Nordisk
Fusaclean, 174t, 178, 192 Natural Plant Protection

G
Galltrol, 170t, 172 AgBiochem Inc.
Product Index 279

Gemstar LC, 150, 15 1 t, 155 Thermo Trilogy


GlioGard, 192 Thermo Trilogy
Granusal, 151t Behring AG
Grey Gold, 183t Eden Bioscience
Gusano, 151 t Thermo Trilogy
Gypchek, 152t USDA Forest Service

I
InCide, 86,87t Crop Genetics International

J
Javelin, 48, 112, 115, 152,255 Thermo Trilogy

K
Kalisena, 175t, 176 IARI
Knockout, 94t Novartis Seeds
Kodiak, 170t, 172, 192, 252 Gustafson

L
Lagniex, 219,220t AgraQuest
Leone, 17 1 t Dominion Biosciences
Lepinox, 884 89, 1 12 Ecogen

M
Madex, 151t Andermatt Biocontrol
Mamestrin, 151t, 238 Natural Plant Protection
Manch, 85, 87t, 1 1 1 Mycogen
"Peril, 85, 88t, 112 Mycogen
"Trek, 247 Mycogen
MVP, 84,85,87, 1 1 I , 115, 1 l5f, 247 Mycogen
Mycostop, 171t, 173, 193 Kemira A g o Oy
Mycotrol -GH, 21 9,220t Mycotech Corporation
Product Index

NatureGard, 94t Mycogen


Naturalis-0, 220t Troy Biosciences
NewLeaf, 92t Monsanto
NewLcaf Plus, 92t Monsanto/ NatureMark
NoGall, 170t, 172 Bio-Care Technology
Norback 84-C, 170t, 172 New Bio Products
Novodor, IO, 80, 106, 107t Abbott (Now Valent BioSciences)

PFR 97,220t Thermo Trilogy


Polygandron, 175, 192 Vys Kumny ustav
Promote, 174t JH Biotech
P.g. suspension, 175t Ecological Labs.

Q
Quantum 4000, 172 Gustafson Inc.

Raven, 83,88t, 134, 134f, 135 Ecogen


Root Shield, 173, 174t Bio Works, Inc.
Rot Stop, 175t, 175 Ecological Labs.

Serenade, 170t, 172 AgraQuest


SoilCard, 174t, 175, 192 USDA
Spherimos, 66t Biochem Products
Spherix, 66t, 69 Biological Preparations
Spodopterin, 15 1t Natural Plant Protection
Spod-X, 150, 151t Thermo Trilogy
Sporeine, 4 International Mineral & Chemlcal
StarLink, 94t AgrEvo
System 3 Seed Treatment, 230 Helena Chemical Co.
Product Index 28 1

Teknar, 57,238 Thermo Trilogy


Thuricide, 4, 9,48t, 114t Thermo Trilogy
Thuringin, 8 Produced in Russia
TM Biocontrol- I , 152t USDA Forest Sevice
T-22 Planter Box, 174t Bioworks
Toarow, 54 Toagosei
Trichodex, 182, 183t, 228,229t Makteshim Chemical Works

VectoBac, 57, 11 1,237 Abbott


VectoLex, 66 Abbott
Vertalec, 220t Tate & Lyle
Victus, 171t, 193 Sylvan Spawn
Virox, 152t USDA Forest Service
Virtuss, 152t USDA Forest Service
VPN, 151t Agricola El Sol

XenTari, 112, 152 Abbott


X-PO, 21 5 Mycogen

Y
Yieldgard, 94t Monsanto
This Page Intentionally Left Blank
Manufacturer Index
(Manufacturer and its products)

A
Abbott Labs. (Now
Valcnt BloSciences),
Dibeta,
DiPel,
DeVinc,
Novodor,
ChicagoIL, USA, 8, IO, 44,48t,52,57, 80, VectoBac FC, VcctoLex,XcnTari
106,107t, 108, I 1 1 , 112, 113, 114, 115, 125,
152, 213t, 214,217,220,234,235,236t, 237,
253
AgBiochem, Inc., Orinda CA, USA, 170t, 172 Galltrol
AgraQuest Corp., Davies CA, USA, 170t, 172, Lagniex, Screnade
2 19, 220t
Agricola El Sol, Brazil, I5 1t VPN
Andermatt Biocontrol, Switzerland, 15 1 t Madex 3
AgrEvo, Wilmington, DE, USA, 94t StarLink

Bayer AG, Germany, 2 19,220t BIO 1020


Becker Microbial Products, Inc., Plantation FL, Aquabac,,, BMP123
USA, 48t, 57, 103, 104t
Behring AG, Werke, Germany, I5 1 t Granusal
BioCare Technology, Somersby, Australia, 170t, Nogall
172
Biochem Products, 66t Spherlmos
Bio-Innovation AL, Toreboda, Sweden, 174t, Binab T
179, 179f
Biological Prepn. Berdesk, Russia, 66t, 69, 103, Bactoculicide, Spherix
104t,
BioWorks Inc., Geneva NY, USA, 173, 174t, T-22, Bio-Trek 22G, Root Shield
191

C
Caffaro S.p.A., CeSano Maderno, Italy, 8 Exobac

283
284 Manufacturer Index

CCT Corpn. Carlsbad CA, USA, 171t, 173, 185t, Deny, Dr. Biosedge
193, 213t, 214

D
Dekalb Genetic Corporation, Dekalb,11, USA, Bt-Xtra
94t
Dominion Bio-Sciences, Inc., Walnut Creeck, Leone
CA, USA, 171t

E
Ecological Labs., Dover, England, 175t, 175 P.g. Suspension, Rot Stop
Ecogen Inc., Langhome PA, USA, IO, 79,79t, Dagger G, Foil, Lepinox, Raven
83, 88t, 89, 1 1 1, 112, 113, 134, 134f, 135, AQ", Aspire, Condor, CryMax,
171t, 172, 183, 183t, 185t, 186, 192,252 Cutlass,
EcoScience , Worcester MA, USA, 185, 185t, BioPath, Bio-Save 1 I , 1 I O & 1000,
192,219, 220t ESC 170GH
EcoSoil System Spot-less
Eden Bioscience, Poulsbo WA, USA, 183t Grey Gold
Encore Technologies, Minnetonka, MN, 213, Collego
213t, 214,217,218,220,253

G
Gustafson Inc., Dallas TX, USA, 170t, 172, 192, Epic, Kodiak
252

H
Helena Chemlcal Co., Memphis, TN, USA, 230 System 3 Seed Treatment

I
IARI, New Delhi, India, 175t, 176 Kalisena
International Mineral & Chemical Corporation, Sporeine
4
Manufacturer index 285

J
JH Biotech, Ventura, CA, USA, 174t Promote

K
Kemira Agro Oy, Helsinki, Finland,171t, 173, Mycostop
193

M
Makteshim Chemical Works, Beer Sheva,Israel, Trichodex
182, 183t, 228,229t
Mauri Foods, North Ryde, Australia, 171t, 172 Conquer
Monsanto Co., St. Louis MO, USA, 92t, 94t, Bollgard, NewLeaf, NewLeaf Plus,
137,138,257 Yield Gard
Mycogen Crop Protection, San Dlego CA, USA, C a s t , Mattch, NatureGard, X-PO,
84, 85, 87, 87t, 88t, 94t, 11 I , 115, 1 l5f, 213, "Peril, MVP, MVP I1
213t, 215,218,247
Mycotech C o p . , Butte MT, USA, 139,219, Mycotrol-GH, BotaniGard
220t, 256

N
Natural Plant Protection S.A.,Nogueres, France, Mamestrin, Spodopterm,
151t, 174t, 178, 192, 238 Caprovirusine, Fusaclean,
New Bio Products, Corvallis, OR, USA, 170t, Norback 84-C
172
Novartis Crop Protection, Greensboro NC, Attribute, Elcar,
USA, 94t, 15 1t, 155
Novo-Nordisk Biochem North America Inc., Foray 48B, Florbac FC
Franklinton NC, USA, 1 1 I , 1 14t

P
Pharmacla & Upjohn, Kalamazoo, MI, USA, Collego
213, 213t, 214, 217,218, 220, 253
Philom Bios. Saskatoon, SK, Canada, 213t. 214, BioMal
21 8,253
286 Index Manufacturer

Plant
Health
Technnologies,
Boise,
Idaho,
BlightBan
A 506
USA, 183t, 193

S & G Seeds,
TheNetherlands, 171
t, 173 BioCoat
Summit Chemical, Baltimore MD, USA, 112, Bactimos briquette
23 8
Stine Microbials, Haskins, KS, USA, 171 t, 173, Deny (formerly Blue Circle)
I85t, 193
Sylvan Spawn, Kittanning, PA, USA, 171t, 193 Victus

Tate and Lyle, UK, 220t Vertalec


Thermo-Trilogy, Columbia MD, USA, 4, 9, IO, Able, Agree, Cyd-X, Delfin,
11,48,48t,57,79,79t,86,87t,lll,112,113, Design, Gemstar LC, GlioGard,
114t, 115, 150, 151t, 152, 155, 174t, 175, 192, Gusano, InCide, Javelin, PFR 97,
220t, 238,255 SoilGard, Spod-X, Teknar,
Thuricide
Tifton Innovation Corp., Tifton, GA, 213t, 214 Dr. Biosedge
Toagosel Co. Ltd., Japan, 54 Toarow
Troy Biosciences, Phoenix, Arizona, USA, 220t Naturalis-0

USDA Forest Service, Otis Air Base, MA, USA, Gypckek, Virox, Virtuss
152t

V
Vyskunny
ustav
rastlinnej,
Piestary,
Slovak
Polygandron
Republic, 175, 192
Pest and
Pathogen Index
Organism (Common name)

Acremoniunt breve, 227 Anagrapha falcifera nucleo


polyhedrovirus (AfNPV), 152, 155
Aedes aegypti (Vector ofYellow
fever), 25, 64, 86, 87t, 88, 104, 108, Androctonus australis (North African
130 scorpion), 159

Aedes triseriatus, 104, I04t Anopheles albimanus, 234


Anopheles culicijhcies. 57, 58t, 66, 66t
Aenalamaria varia. 220t Anopheles stephensi (Vector of
Malaria), 57, 58t, 66, 66t, 130
Aeschynonlene virginica (Northern Anopheles subpictus, 66
joint vetch -weed), 201t
Anticarsia gemmatalis (Velvetbean
Agelastica altti (Alder leaf beetle),44 caterpillar), 46t, 148
Anticarsia genvnatalis nucleopoly-
Agrobacteriunl radiobacter hedro virus (AgNPV), 148, 151t,
(Saprophyte of legume), 170t, 172 155, 157
Agrobacteriunl turnefaciens (Tumorous
crowngalls on dicotyledonous plants Argyrotaerua spp. (Tortnx moth), 46t
- pathogen), 90, 170t
Arndlaria nlellea (Root rot of woody
Agrotis ipsilon (Black cutworm), 9 hosts), 174t

Alternaria brassicicola (Leaf spot & Aschersonia aleyrodis, 2 12


blight in crucifers - pathogen), 171t
Alternaria cassiae (Foliar blight- Ascochyta caulina (Ascochyta blight-
Inducing pathogen), 201 t, 203,206, pathogen), 203
232t, 233
Aspergillus niger (Black mold -
Ampelontyces quisqualis(Hyper- pathogen), 174t, 175
parasite of powderymildews), 173,
182t, 183, 192,252 Autographa ca[ifornica (Alfalfa
looper), 155, 157, 163, 251
Anagasta (Ephestia) kuehniella Autographa callfornica nucleo-
(Mediterranean flour moth), 24,44 polyhedrovirus (AcNPV), 15 1, 15 1t,
157, 163,239,254
Anagrapha falcifera (Celery looper),
152

287
288 Pest and Pathogen Index

-thompsoni. 9
-1huringiensrs. 8. 9. 19. 34
Bacillus anthralis. 3. 30 -tolworthi, 19
Bacillus brevis, 227, 229, 229t
Bacillus cereus. 5. 9, 166 Beauveria bussiana (White muscardine
Bacillus laterosporous, 5 disease - pathogen), 21 2.2 13f. 2 15.
Bacillus licheniformis, 227 216. 217. 218, 219,. 220t.239.240.
Bacillus megaterium (Cotton and okra 256
leaf colonizing bacterium),85. 87t. Beauveria brongniartii.
183t
Bacillus polynyxa. 227 Bombyx mori (Silk worm), 19, 27f, 34.
Bacilluspopilliae (Milky diseasein 161
scarabaeid grubs). 5 Bornbyx mori nucleopolyhedrovirus
Bacillus sphaericus. 43. 60, 61 f. 62. (BmNPV), 157
62f. 63.64, 65t, 66t(2). 70t, 86, 87t.
132 Botrytis cinerea (Gray mold in grapes.
Bacillus subtilis (Frenching in tobacco tomatoes and potted plants
- pathogen), 170t, 172. 192, 229t. pathogen), 176f. 181, 182,182t. 185,
230,230f 185t. 188.226,228. 229t
Bacillus thuringiensis. 3. 6f. 9, 45f. 49f, Bottytis septoria, 171 t
90. 1 14. 134,254
-alesti, 9, 34 Bromus tectorum (Downy brome-
-oizawal. 9, 14. 25.44, 79t. 80. pathogen). 203
83f. 87t. 126, 127, 128. 134
-herliner, 4. 106. 108 Burkholderia (Pseudomonas)ccpucia
-canadensis, 8, 56 (Sour skinof onion - pathogen). 85.
-darmstadiensis. 20. 56 17lt. 172, 185t. 192. 227
-finitinus. 9
-jukuskaensis. 20
-galleriae, 8. 56 C
-isruelensis, 4, 15, 20, 24, 43, 54.
70t. 86, 88. 101, 104, 130. 130f, Cadra cuutella (Almond moth). I25
130t. 131t. 132. 237f
yegathesun. 20. 56 Candida oleophila. 185, 185t. 192
-kenycre. 9. 19
-kumarnotoensis. 8 Cassra (Senna) obtusijolia (Sicklepod -
-kurstaki. 43, 44, 45f, 47t,48t. weed), 20 1. 20 1 t. 232t
49f, 79, 80. 83. 85, 86, 88t. 101,
115, 124,125.126.128. 134 Caulobacter crescentus. 86.87t
-kyushuensi, 20, 56
-medellin. 56
Ceratocystis ulmi (Dutch elm disease
-morrisoni. 8, 14, 15. 20.44. 56,
of Elm - pathogen), 174t
-shandongiensis, 56
-sun diego, I9,44
Cercospora rodmonii (Leafspot -
-sotto. 4. 9. 34. 45
pathogen), 232t
-tenebrionis. 4. 14. 16. 19, 20, 44.
79. 79t. 86, 87t. 91, 106, 107,
107t
Pest and Pathogen Index 289

Clrenopodiutn album (Annual Coniotlryriunzminitans, 180


herbaceous - weed), 203
Culexpipiens (Vector of bancroftian
Clzilo suppressalis (Rice stemborer), 95 filariasis), 63, 130
Clrondrostereurn purpureunz (Silver Culex quinquefascialus, (Filariasis
leaf of plum and apple - pathogen), vector), 58t, 87t, 88, 130, 131, 132
174t Culex tarsalis, 238

Clzoristoneura funliferarm(Spruce Culicrrronzycesclavosponcs, 21 3,219


budworm), 24,46t, 112,236
Clzoristoneura occiderztalis(Western Culiseta incidens, 60
spruce budworm), 50
Cydia pomonella, (Codling moth), 45
Clrrysomela scripta (Cottonwood leaf Cydia ponronellagranulovirus (CpGV),
beetle), 20,44 151t

Cirsiutn arvetzse(Canada thistle - Cyperus spp. (Nutsedges - weeds), 202,


weed), 208 203
Cyperus esculentus(Yellow nutsedge -
Clavibacter xyli subsp. cynodontis, weed), 201t, 202,203
(Xylem-inhabiting endophyte Cyperus irra (Rice flat sedge -weed),
bacterium), 86, 87t 203
Cyperus rotundus(Purple nutsedge -
Cnaplzalocrosis nledinalis,(Rice leaf weed), 203
folder/roller), 95

Cochliobolus (Curvularia) lunatus,


232t
Dactylaria higginsii (Lutterell)
Colletotrichrcnr coccodes (Potato black (Phytopathogen for control of
dot and tomato rootrot - pathogen), nutsedges weeds), 203
232t, 233
Colletotrichutn gloeosporioides Diaprepes abbreviatus (Citrus root
(Wither tip in citrus, mango weevil - pathogen), 239
anthracnose - pathogen), 232t
Colletotrichutn gloeosporrordesf. sp. Dictyosporium elegans, 180
aeschynon1ene (Anthracnose-inciting
pathogen of northen Joint vetch), Diparopsis watersi (Red bollworm or
20 1t, 206 Sudan bollworm), 238
Colletotrichunz gloeosporioidesf. sp.
rnalvae (Anthracnose-causing
pathogen of round-leaf mallow), E
20 1t, 202
Colletotrichun~orbiculare, 207
Colletotrichum truncatutn (Pathogen of Endothia parasitica (Chestnut blight in
weed hemp sesbania), 205,206,233 chestnut - pathogen), 174t
290 Pest and Pathogen Index

Enterobacter aerogerres, 170t, 172, Fusariunt solanz, (Root rot - pathogen),


229, 229t 175t, 176, 188
Fusarium solani f. sp. cucurbitae (Root
Entomophaga nraimaiga,(Entomo- rot in cucurbit - pathogen), 232t
pathogen fungus of Lymantria
dispar), 212

Entonzophthora muscae.2 12
G
Gaeunmrnomyces graminis var. tritici
Ephestia (Cadra) cautella (Almond (Seedling disease& Take-all disease
moth), 4 , 4 4 of wheat and other cereals-
pathogen), 189, 190
Erwinia amylovora (Fire blight and
frost injury in pear & apple -
Gallerza nzellonella(Greater Wax
pathogen), 193
moth), 24
Erwlnia herbicola, 181
Etysiphe cichoracearum (Powdery
Geotnchum candidunr (Citrus sour rot-
mildew in cucurbits, okra, tobacco
pathogen), 185t
pathogen), 183t
Gliocladium resewn, 182
Escherichia coli, 82, 158
Gliocladium (Trichodernm) virens,
173, 173t, 175, 180, 186, 191,227,
Estigrrzene acraea (Saltmarsh
228,229t
caterpillar), 46t

Exophiala jeanselnri, 186

F Helicoverpa spp. (Bollworm,


budworm), 46t
Helicoverpa armigera (Amerlcan
Fusarium avenaceunz (Root rot & pre- cotton bollworm), 234,235,238
emergence damping in lentil -
Helicoverpa punctigera (Tobacco
pathogen), 229t, 230,230f
budworm), 235
Fusarrunr granrinearum (Gibberella
zeae) (Scab in wheat - pathogen),
Heliothis spp. (Budworm, fruitworm,
188
bollworm), 44,45,46t
Fusarium lateritiunr,232t, 233 Heliothis armigera (Tomato budworm/
Fusarium moniliforme (Gibberella fruitworm), 149
fujikuroi) (Fusarium root and stalk
Heliothis virescens (Tobacco budworm
rot of maize, sorghum- pathogen), or cotton boll worm), 53, 83,90,91,
178
105, 113, 119, 125, 133, 137, 139t,
Fusarium oxysporunz (Fusarium wilt - 140,238,239,257
pathogen), 176, 177, 177f, 178, 190, Heliothis (Helicoverpa) zea (Corn
192,227 earworm or bollworm), 9; 83, 91,
Fusarium oxysporum f. sp. dianthi 113, 133, 137, 139, 149,257
(Carnation wilt - pathogen), 171t,
HeliothdHelicoverpa nucleopoly-
173t hedrovirus (HzNPV), 15 1t, 154, 155
Fusarlunr proliforatum, 183
Pest and Pathogen Index 291

Heterocampa maneto (Oak leaf "lava pusilla Sm (Round-leaved


caterpillar). 53 mallow - weed). 201t, 202
Heterobasrdion annosum. (Cabbage Marnestra brassicae (Cabbage moth).
boredpine stem and rootrot - 471, 135
pathogen), 1741. 175 Mamestra brossicae nucleopolyhedro-
virus (MbNPV), 1511, 238
Ilirsutella thompsonii (Aacro-
Pathogenic fungus). 2 12 Manduca sextu (Tobacco hornworm),
46t, 89,90. 135
Hyphantrra cunea (Fall webworm). 461 Manduca spp. (Hornworm). 471

Mansonia spp., 701

Muruca testulalis (Leguminous pod


borer and leaf webber). 238
Lagenidium giganteum, 2 13, 2 19, 220t
Metnrhizium unisopliae (Green
Leirus quinquestriatus lebraeus muscardinc disease - pathogen), 2 12.
(Yellow scorpion). 160 213f. 214f. 215,218.219.220t.236
Metarhr=iumjluvoviride. (Entomo-
Leptinotarsa decemlineata (Colarodo pathogenic fungus), 21 2. 2 15. 2 17.
potato beetle), 44. 84, 91 239
Lobesia botrana, (Grape vine moth). Morrenia odorata (Strangler vine or
471 milkweed vine - weed) 201t. 202.
205
Lymantriu dispar (Gypsy moth), 24.
471, 105, 112, 126, 129, 212 Mucor piriformis (Muscor rot
Lvmantria dispar nucleopolyhedro- pathogen), 1851
virus (LdNPV). 149. 1521. 153. 155.
239 Mycocentrospora acerina, 188

Macocentrospora acerrna. 189


Neodiprron sertfer (European pine
Macrophomina spp. (Charcoal rot or sawfly), 152t
dry rot in millet - pathogen). 169 Neodiprron sertrfernucleopolyhedro-
Macrophomina phaseolina (Charcoal virus (NeSeNPV). 152t
rot of soybean/ dry root rot of mung
bean -pathogen), 174t Nomuraea rileyr (Entomogenous
fungus ofAnticarsia gemrnatnlis).
Malacosoma spp. (Tent caterpillars), 212
471
Pest and Pathogen Index

Phymatotrichum omnivonrm (Root rot


-
of cotton and other leaf plants
Orgyia pseudotsugata (Douglas fir pathogen), 200
tussock moth), 152t
Orgyla pseudosugata nucleopoly- Phytophthora cactorutn (Root rot of
hedrovirus (OpNPV), 152t ginseng and crown and root rot of
apple trees - pathogen), 170t, 172,
Otyctes rhinoceros (Coconut 178, 179f, 229,229t
Phytophthora capsici (Phytophthora
rhinoceros beetle), 148
Otyctes baculovirus, 148 blight of pepper- pathogen), 227
Phytophthora cinnatnonli (Root-rotting
Ostrinia nubilalis (European corn of numerous woody plants-
borer), 47t, 92, 1 14, 139 pathogen), 179
Phytophthora megaspertna (Damping
Otiorhynchus sulcatus (Black vine off & root rot in alfalfa and foot rot
weevil), 21 9,220t in soybean - pathogen), 232t
Phytophthora palnrlvora (Bud rots of
palm, damping off in okra, tobacco
and seedling disease of papaya-
pathogen), 179,202, 201t, 205,253

Paecilotnyces farinosus, 2 12, 15


2 Pieris brassicae (European cabbage
Paecilotnycesfitmosoroseus (Entomo- butterfly), 24,25,49, 80
pathogenic fungus), 212,215,220t Piens brassicae granulosis virus
(PbGV), 238
Paropsis chatybdis (Eucalyptus
tortoise beetle), 44 Platiphena scabra (Green
cloverworm), 47t
Pectinophora gossypiella (Pink
bollworm), 44,91, 234, 257 Platynota stultana (Omnivorous leaf
roller), 47t
Penicillium citrinutn, 180
Penicilliutn digitatunl (Green mold), Plodia interpunctella (Indian meal
185, 185t moth), 124, 125, 126f
Penicilliunz expansurn (Blue-mold rot
on pear, apple & citrus - pathogen), Plutella xylostella (Diamondback
185, 1 8 9 moth), 24,47t, 83, 118, 125, 133,
134, 138, 140,238
Penicillium italicunl (Blue mold), 185
Poa annua (Annual bluegrass - weed),
Peniophora gigantea, 175t 203

Phologophora tnetuculosa (Angle Pseudonlonasfluorescens (Root


shade moth), 47t colonizing bacterium), 84, SSf, 87t,
88t, 115, 170t, 172, I82t, 189, 192,
Phthorunaea operculella (Potato tuber 203
moth / tobacco leaf miner), 9I , 238 Pseudomonas putida, 189
Pest and Pathogen Index 293

Pseudomonas (Burkholderia)
solanacearunl (Bacterial wilt disease
in brinjal, potatoes &bacterial brown Sclerotinia homeocarpa (Brown patch
rot of potato- pathogen), 200 of turf and dollar spotin blue grass -
Pseudonronas syringae (Brown spot in pathogen), 174t
lima bean - pathogen), 182t, 185, Sclerotinia minor (Lettuce drop -
192 pathogen), 180, 181
Pseudonlonas syringae pv. Tagetis, Sclerotirlia sclerotiorunl (Blight & dry
203,208 rot in vegetables and ornamental
Pseudomonas tolassii (Bacterial blotch plants - pathogen), 175t, 176, 180,
of mushroom - pathogen), 171t, 172, 200
192 Sclerotinia trifoliorum (Crown rot of
Pseudoplusia includens (Soybean clover -pathogen), 180
looper), 1 14
Sclerotiunl(Corticium) rolfsii(Leaf and
Psorophora spp., 57 foot rot disease and Southern blight
on tomato and pepper- pathogen),
Puccinia canaliculata, 201 t,203 173t, 175,200,227
Pyemotes tritici (Straw Itch mite), 1GO Septoria spp. (Leaf spot diseaseon
cereals - pathogen), 171t
PytAiunl nunn, 175 Sesbailia exaltata (Hemp sesbania -
Pythiunl oligandrunl (Damping offin weed), 206,207t
corn, bean, vegetables etc. -
pathogen), 174t, 175, 176f, 192
Sinluliunl danmosum (Vector of river
Pythiunl ultirnunl (Damping off of
blindness), 55, 71
cotton seedling disease/ root rot of
wheat - pathogen), 170t, 175, 18 I , Sinlulium vittatunl, 52
189, 190, 191,200,227,229
Pythiunl vexam, 175 Sitona hispidulus (Clover root
curculio), 86

Sitona lineatus (Pea leaf weevil), 86

Sphaerotheca fuliginea (Powdery


Rhizobium Ieguminosarunt B V vicaea,
mildew in cucumber - pathogen),
86
181, 182
Rhnobiunl nleliloti, 86, 87t
Rhizoctonia carotae, 189
Rhizoctonia solani (Damping off, stem Spodoptera spp. (Army worms), 19,47t
canker, storage rot, sheath blight - Spodoptera exigua (Beet armyworm),
pathogen), 170t, 171t, 173t, 175, 9, 24, 83, 126, 135, 139,
187f, 189, 19 I , 227,232t Spodoptera exigua nucleopolyhedro-
virus (SeNPV), 15 1t
Rhodotorula glutinis (Russet of apple- Spodoptera frugiperda (Fall army-
pathogen), 182t worm), 9, 149, 161, 238, 7, 146, 157,
23 I
Pest and Pathogen Index

Spodoptera littoralis (Cotton Trichoplusia ni (Cabbage looper), 47t,


leafworm), 14, 84, 108, 127, 127f, 83, 108, 118, 163
149,238 Trrchophia ni multinucleopolyhedro-
Spodoptera littoralis nucleopolyhedro- virus (TnMNPV), 163
virus (SINPV), 15 1 t Trichophia ni Granulovirus (TnGV),
163
Sporidesmium sclerotivorum, 180

Sporothrixflucculosa, 183 U
Streptomyces grrseoviridis (Root
colonizing actinomycetes), 171 t, Udeaa ferrugalis (Soybean leaf trier),
47t
173,192
Uncinula necator (Powdery mildew in
grapevine grown in green house -
T pathogen), 181, 182, 182t, 188

Talaromycesflavus, 1 SO Ustilago avenue (Loose smut of oats


-
pathogen), 188
Teratosperma oligocladium, 180

Tllielaviopsis spp. (Black root rot -


pathogen), 171 t V
Tolypocladiurn cylindrosporum, 2 13 Verticillium lecanii, 183, 212, 217,
220t
Tortrix spp. (Leaffolders), 47t Verticillium malthousei, 1 7 4

Trichoderma l~amatum,182t, 186


Trichoderma hanianum, 173, 174t, X
175, 182, 182t, 186, 187f, 188,228,
229t, 252 Xanthomonas campestris (Black rot in
Trichoderma polysporum, 174t crucifers - pathogen), 203
Subject Index

Aerial plant diseases, 182 Formulation, 13


Control of, I8 I , 183t Commercial transconjugate
Grey mold, 182 products, 79t
Powdcry mildews, 182 Genetically modified strains
Agrobacteriunl tumefacietts of, 77
mediated transformation, 90,91 f Gene types, cry, 19,20
Antagonism, 167 History, 4
Antagonists, microbial, 167, 169f, 17% Insecticidal Activity, 21
174t, 176f, 183t, 185t Variation, 22
Antibiosis, 168, 184 Determination of, 22
Life cycle, 7, 7f
Bacillus sphaericus, 60 Mode of action, 24, 27f
Characteristics, 6 1, 6 1f, 62f Mechanism, 25
Classification, 63,65t Bivalent two-step binding
Commcrclal products, 66t model, 30
Comparlson with Bti, 69, 70t Penknife model, 30, 29f
Insecticidal activity, 64 Umbrella model, 28,29f
Temperature effect,66,66t, Morphology of parasporal
67f inclusions, 15, 15f
Mode of action, 67 Occurrence, 5
Persistence, 68 Persistence, 30
Resistance, 132 Factors affecting, 31, 32t
Safety and ecotoxicology, 69 Protein Engineering, 89
Transgenic, 87t Production, 9
Toxins produced, 62 Fermentation, 1 1
Binary, 63 Semi-solid, 12
Mosquitocidal, 63 Submerged, 12, 13f
Resistance in Lepidoptera,
Bacillus thuringiensis (Bi),3 124, 125f, 126f, 127f
Bioassay, 23 Estimation of, 138
Characteristics, 5 , 6 f Monitoring of, 133
Classification and nomenclature, Resistance management, 132
13 By designing recombinant
Commercialization, 254 Bt strain, 134, 134f, 135f
Crystal proteins and genes, 16 General strategies, 133
Classification and In transgenic plants, 136,
nomenclature, 16, 17t(2), 136f, 137, 138
1s t Safety and ecotoxlcology,32
Structure, 2 0 , 2 1f Beneficial insects,
Cry-Cry interaction, 129, 129f, affect on, 34
134 Mammalian safety, 33
Encoding protoxins, 18 Non-target organisms.
affect on, 34
295
Subject Index

Toxiclty to avian and fish, 33 Non-target organisms, affect


Spore on, 60
Count, 24 Synerglstic combination of, 237,
Crystal Intcractlons, 126, 127f (2) 237f, 238
Role In tox~c~ty, 23 Synergistic interaction of toxin,
127f (2) 130, 130f, 131t(2)
Strain
Genetically modified,77 Bt-kurstaki, 44
Novel Bt through Characteristics, 44
conventional genetic Commercial products, 48t
techniques, 78 Crystal proteins and genes, 45
Selection, I O Effect on beneficial insects, 53
Serotyping, 14 Effect on non-target organisms, 52
Toxins produced, 7 Effect on parasites, 52
a-exotoxin, 8 Effect on predators, 53
p-exotoxin, 8, 8f Encoding genes, 45
6-endotoxin, 8 , 2 1 f Insecticidal activity, 46,46t, 47t
Vegetative insecticidal protelns, 9 Mammalian safety, 5 1
Transgenic cotton, 91f, 92t, 137, Mode of action,48,49f
257,258f Parasporal crystal, 45,45f
Transgenic micro organisms, 84, Persistence, 49
85f, 87t Factor effecting, 50
Transgenic plants, 77, 90 Safety and ecotoxicological
Bt-corn, 94f, 94t, 134 effects, 5 1
Bt-cotton, 91 f, 92t, 257,258f Toxicity and pathogenecity to
Bt-Potato, 92t fish and bird, 52
Commercialization of, 257, Synergistlc comblnatlons of,
258f 234,235f, 236t, 236
Dlcotyledonous, 90,91 f, 92t
Monocotyledonous, 92, 94f, Bacterial biofungicides, 169f, 170, I70t
94t Bacterial bioherbicides, 203
Second generation of, 95 Bacterial insecticides, 3
for crop and forest protectlon, 43
Bt-israelensis, 54 for insect vector control, 43
Characteristlcs, 55
Comparison with Bs, 69,70t Baculoviruses, 147, 149f
Crystal proteins and genes, 55, 56f Characteristics, 149
Effect on resistance develop-ment, Classification, 149
130, 13Of, 131t(2) Commercialization of, 255
Encoding genes, 56 Commercial products, NPVs, 150,
Insecticidal activity, 57 151t, 152t
Factor affecting, 57,58t(2) Enhancement of actwity by
Mode of action,58 Enhancin, 156, 156f
Parasporal inclusions, 55 Formulation, 155
Persistence, 59 Mode of action, 152, 153f, 154f
Safety and ecotoxicological Production, I54
effects, 59 Recombinant, 157, I58f, 159t
Mammalian Safety, 59 Deletion of egt genes, 161
Subject Index 297

Environment risk assessment, Integrated use of, 232


162 Production, 205
Expressing toxin, 159 Rationale of synergistic
Insect hormones, 160 combinations, 231
Juvenile hormone esterase, Synergistic interactions with
161 chemical herbicides, 232,
Synergistic combination with 232t, 233f
synthetic insecticides, 238
Bioassay of microbial insecticides, 23 Bioinsecticides,
Commercialization of, 253
Biofungicides, 167, 168f, 169f Fungal, 21 1
Bacterial and actinomycetous, Integrated use of, 234
170, 170t Biopesticides,
Commercialization of, 250,25 1f Commercialization of, 245
Case studies, 252 Fast track registration of, 246
Control of aerial plant diseases, Integrated use of, 225
181, 182t Market environment of, 249
Control of postharvest decay, 184, Search for suitable model,
185t 250
Control of soil borne diseases, Technical constraints, 248
169, 170t, 174t, 175f Genetic techniques for strain
Fluorescent pseudomonads, improvement, 78
biological control with, 177f, Classical mutation, 80
189 Conjugation, 78,79t
Formulation of, 191 Electroporatlon, 80
Fungal, 173, 174t Transduction, 80
Integrated use of, 226
Bacterial antagonists, 226 Encapsulation ofB f ,
Biofungicides with chemical Cellcap, 85, 85f, 115, 115f
fungicides, 228,229t, 230f Matricap, 1 15
Fungal and bacterial Starch matrix, 1 14
antagonists, 227
Mycoparasites, 175, 176f, 180f, Formulation o f B f , 13, 101
181 Attributes, 103
Production of, 190 Feeding stimulants, 105,
Trichodernm spp., biological IOSf, 106f, 107t
control with, 186, 187f, 188f Non-toxic/mildly toxic
additives, 108, 109f
Bioherbicides, 199 Particle size, I03,104t(2)
Bacterial, 203 Characteristics, 102
Formulation of, 207 Commonly used, 11 1
Synergistic interactions with Effect of adjuvants, 109
chemical herbicide, 233 Effectiveness at low dose rate, 1 18
Commercialization of, 252 Improved, 1 12
Environmental limitation of Encapsulated, 1 14, 1 I Sf
efficacy, 204,204f Oil-based ES, 113, 113f, I14t
Fungal, 200,201t Synergism by Enhancin, 109
Formulation of, 205,207f
298 Subject Index

Target specific tailor made, 1 16, Insecticidal activity, 19


117f B. sphaericus of, 64
Formulation of Bt of, 20
Bacterial Insecticides, 101 Bti of, 57
Baculoviruses, 155 B f k of, 46, 108
Bacterial antagonists, 19 1 Determination of, 22
Biofungicides, 19 1 Spectra, 139t
Fungal antagonists, 187 Variation of Bt. 22
Fungal bioherbicides, 205, 207f
Invert emulsion formulation, Integrated use of
206,207f Biofungicides, 226
Oil-based suspension Bioherbicides, 231
emulsions, 207 Bioinsecticlde, 234
Fungal bioinsecticides, 217, 218f Biopesticide and
Fungal bioinsecticides, 21 1 synthetic pesticides, 225
Fungal spores
Dormancy, 21 7 Microbial insecticides
Hydrophobicity, 2 17 Bacterial, 3, 43
Post application longevity, 219 Bioassay of, 23
F1tsunum wilts, 177, 177f Characteristics, 102
Formulation of, 101
Genetic engineering, 8 I , 81 f, 82f Fungal (Myco-), 21 1
Genetic modificatlon Integrated use, 225
Bacillus sphaericus, 86 Viral (Baculoviruses), 147
Baculovirus, 157, ISSf, 159t
Bt-strains, 77 Mycoinsecticides, 2I 1
Crop plants, insect tolerant,89, Commercial, 219,220t
92t Commercialization